OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

FOREWORDINTRODUCTION

2,6-di-tert-butyl-p-cresol (BHT)
CAS N°:128-37-0

UNEP PUBLICATIONS

1OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

SIDS Initial Assessment Report
For
SIAM 14
Paris, France, 26-28 March 2002

1. Chemical Name:

2,6-di-tert-butyl-p-cresol (BHT)

2. CAS Number:

128-37-0

3. Sponsor Country:

Germany

4. Shared Partnership with:
5. Roles/Responsibilities of
the Partners:
Name of industry sponsor/consortium•

BMU (Bundesministerium für Umwelt, Naturschutz undReaktorsicherheit)
Contact person:
Address:

Prof. Dr. Ulrich Schlottmann
Postfach 12 06 29
D- 53048 Bonn- Bad Godesberg

Process used•

6. Sponsorship History
How was the chemical or
category brought into theOECD HPV Chemicals
Programme ?

•see next page

7. Review Process Prior to
the SIAM:
8. Quality check process:No new SIDS testing ( X )New SIDS testing( )9. Date of Submission:

01 February 2002

10. Date of last Update:last literature research:
Toxicology:14.07.01 Ecotoxicology: 16.07.01

11. Comments:*

OECD/ICCA - The BUA* Peer Review Process

BUA (GDCh-Beratergremium für Altstoffe):
Association of German Chemists (GDCh)

2

Advisory

Committee

UNEP PUBLICATIONSon

Existing

ChemicalsoftheOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
Qualified BUA personnel (toxicologists, ecotoxicologists)
perform a quality control on the full SIDS dossier submitted byindustry. This quality control process follows internal BUA
guidelines/instructions for the OECD/ICCA peer review process
and includes:-a full (or update) literature search to verify completenessof data provided by industry in the IUCLID/HEDSET-

Review of data and assessment of the quality of data-

Review of data evaluation-

Check of adequacy of selection process for key studies
for OECD endpoints, and, where relevant, for non-OECD
endpoints by checking original reports/publications-

Review of key study description according robust
summaries requirements; completeness and correctness ischeckedagainstoriginal
reports/publications(if original reports are missing: reliability (4) not
assignable)-

Review of validity of structure-activity relationships-

Review of full SIDS dossier (including SIAR, SIAP andproposal for conclusion and recommendation for furtherwork)

In case of data gaps, review of testing plan or rationale for not
testing.

UNEP PUBLICATIONS

3

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
SIDS INITIAL ASSESSMENT PROFILECAS No.

128-37-0

Chemical Name

2,6-di-tert.-butyl-p-cresol (BHT)
Butylated HydroxytolueneOHt -C4H9t -C4H9
Structural Formula

CH3
RECOMMENDATIONS
The chemical is a candidate for further work.

SUMMARY CONCLUSIONS OF THE SIAR
Human Health
BHT is of low acute toxicity. BHT caused acute toxic effects in mammalians but there were no specific clinicalsymptoms. In rats, the oral LD50 was > 2930 mg/kg bw, the LD50 after dermal exposure was > 2000 mg/kg bw. It
was slightly irritating to the skin and eyes of rabbits.
On chronic oral exposure of rats, liver and thyroid are the main targets. Doses above 25 mg/kg bw/day BHT resulted
in thyroid hyperactivity, enlargement of the liver, induction of several liver enzymes. 25 mg/kg bw/day BHT can beconsidered as NOAEL for chronic exposure. The haemorrhagic effects of high repeated doses of BHT seen incertain strains of mice and rats, but not in other species, may be related to its ability to interact with prothrombin andvitamin K.
BHT showed no potential to cause point mutations in several bacterial and mammalian in vitro test systems.
Overall, the available studies demonstrate that BHT has no clastogenic activity in vitro or in vivo. Most in vitro
chromosome aberration assays were negative as were sister chromatid exchange assays and DNA damage and repair
assays. In vivo, micronucleus assays with mice, cytogenetic assays with rats and mice, dominant lethal assays withrats and mice, and the heritable translocation assay with mice were also negative.
BHT is not a genotoxic carcinogen. Carcinogenic effects observed in one long-term study with rats probably werecaused by the specific study conditions. However, it cannot be completely ruled out that the hepatotoxic effects
caused by high and chronic doses of BHT may result in persistent cell proliferation, which is known as a possible
mechanism of non-genotoxic carcinogens. In addition, depending on the application regime, BHT may exert either
anticarcinogenic or tumour-promoting activity at relatively high doses. For the possible carcinogenic and tumourpromoting effect of BHT, a threshold level of 100 mg/kg bw/day can be assumed. At this dose, no increase in theincidence of liver carcinoma, but a slight increase in liver adenoma were observed after chronic exposure starting inutero as a worst case scenario.
The only effects on reproduction were lower numbers of litters of ten or more pups at birth at doses of 100 mg/kg
bw/day and above. The NOAEL was 25 mg/kg bw/day.
From studies with mice and rats there is no evidence of teratogenic effects of BHT. During pregnancy BHT had
maternal effects on mice above oral doses of 240 mg/kg bw/day. The NOEL for developmental toxicity was 800
mg/kg bw day.
Despite of being in wide dispersive use as ingredient of various products for many years only very few cases of

4

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

allergic reaction in humans after dermal exposure or oral intake have been described. For the use of BHT as
antioxidant in foodstuff an acceptable daily intake (ADI) of 0 - 0.3 mg/kg bw/day has been established.
Environment
BHT has a melting point of ca. 70 °C, a water solubility in the range of 0.6-1.1 mg/l (20-25 °C), a density of 1.03
g/cm³, and a vapor pressure of 1.1 Pa (20 °C). The measured log Kow is determined to be 5.1.
According to a Mackay Level I model calculation, the main target compartment for BHT is air (79-87 %), followedby soil (6.1-10.2 %) and sediment (5.7-9.5 %). Due to the instability of BHT in aqueous solution the estimations
reflect a tendency for BHT distribution among environmental compartments. BHT is relatively unstable underenvironmental conditions. Extent and products of decomposition are dependent on several factors like irradiation,
pH, temperature, moisture, presence of soil and soil microorganisms, and oxygen content. In air BHT is indirectly
photodegradable by hydroxyl radicals with t1/2 = 7.0 hours. In aqueous solution BHT is decomposed in natural
sunlight with irradiation (ca. 75 %) and without (ca. 40 %), forming different, partly unidentified metabolites. BHT
is also not stable in soil. Within one day of incubation 63-82 % of BHT were decomposed in non-sterilized and 2535 % in sterilized soils. A mineralization up to 30 % was observed under non-sterilized conditions. Depending on
the exposure pathways, the compartments air, hydrosphere and soil can be environmental target compartments forthis substance and its metabolites. BHT is not readily biodegradable in water according to a modified MITI-I test
(4.5 % degradation after 28 days). A wide range of bioconcentration factors (BCF) was found in different
experiments. Bioconcentration factors (BCF) in the range of 230-2500 have been determined for fish after 56 days.
The BCF values determined after a 28 days exposure period in a model ecosystem with soil were 2-17 for fish, 30
for snails and 38 for algae. It can be assumed that BHT has a moderate to high bioaccumulation potential in aquatic
species.
For the toxicity of BHT on aquatic species reliable experimental results from tests with fish, daphnia, and algae areavailable. Only those effect values are considered for the assessment that did not exceed the low water solubility ofBHT (0.6 - 1.1 mg/l) and were based on measured concentrations. The lowest reliable acute toxicity values are:fish (Brachydanio rerio): 96h LC0 ≥ 0.57 mg/l;
invertebrates (Daphnia magna): 48h EC0 ≥ 0.17 mg/l;
algae (Scenedesmus subspicatus): 72h ErC8 = 0.4 mg/l. This value can be used as a NOEC.
In a 21 days reproduction test with Daphnia magna a NOEC = 0.07 mg/l was determined. Using an assessment
factor of 50,a PNECaqua = 0.0014 mg/l is derived from this long term NOEC.
Exposure
In 2000, the world production capacity of BHT amounts to about 62,000 t/a by more than 20 producers. BHT is a
registered antioxydant, licenced for food products, animal feed, cosmetics, and packaging material. It is also used inpetroleum products, synthetic rubbers, plastics, elastomers, oils, waxes, soaps, paints, and inks.
Releases into the environment may occur during production of BHT as well as during its use in different
applications as stabilizer and during the use of the products that contain the substance. A significant release into theenvironment is expected from migration of BHT onto the surface of products containing the substance.

NATURE OF FURTHER WORK RECOMMENDED
Environment: The substance is a candidate for further work. Releases into the environment during use of BHT andfrom products containing the substance have to be assumed but are not quantifiable. In the environment, BHT is
rapidly decomposed forming several, partly unidentified, metabolites. BHT is not readily biodegradable, a moderate
to high bioaccumulation potential has to be assumed. The NOEC from the long-term toxicity to daphnids was 0.07
mg/l, resulting in a PNEC of 0.0014 mg/l. Therefore, the performance of an environmental risk assessment isrecommended. Especially the questions concerning exposure, bioaccumulation as well as toxicity of the metabolites
should be clarified.
Human Health: No recommendation for further work, because all SIDS endpoints are adequately covered andbecause exposure is controlled in occupational settings.

UNEP PUBLICATIONS

5OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

SIDS Initial Assessment Report
1

IDENTITY

1.1

Identification of the Substance

CAS Number:
IUPAC Name:
Molecular Formula:
Structural Formula:128-37-0
2,6-di-tert-butyl-p-cresol (BHT)
C15H24OOH

t -C4H9t -C4H9CH3

1.2

Physico-Chemical properties

At room temperature BHT is a colorless solid, the melting point is ca. 70 °C (Pardee 1944; Chang& Maurey 1985). The density of BHT is given with 1.03 g/cm³ (Bayer AG 1973), the vapour
pressure of the substance is 1.1 Pa (Bayer AG 1986a). Both values relate to 20 °C. The measured
log Kow is determined to be 5.1 (Shell Research Ltd. 1983).
The determination of the water solubility is complicated by the fact that BHT has a very low
solubility, older literature state "not soluble in water" and that BHT reacts with oxygen (see chapter
2.1.2). According to the available data the water solubility is in the range of 0.6 to 1.1 mg/l at 20 25 °C (Inui 1979a, Bayer AG 1986b) and about 1.5 mg/l at 30 °C (extrapolated value; Chang &Maurey 1985).
The purity of BHT produced by Bayer AG is given with > 99.8 % w/w (Bayer AG 2001).

6

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

2

GENERAL INFORMATION ON EXPOSURE

2.1

Production Volumes and Use Pattern

In 2000, the world production capacity of BHT amounted to about 62,000 t/a. This figure can be
split up as follows:
USA, 2 producers
Japan, 3 producers
Western Europe, 4 producers
RussiaIndia
China, 8 producers

about 7,000 t/a
about 15,000 t/a
about 25,000 t/a
about 5,000 t/a
about 1,000 t/a
about 9,000 t/a

2,6-Di-tert-butyl-p-cresol (BHT) can be produced by dibutylation of p-cresol or the butylation in
two steps of a m-/p-cresol mixture including the separation of BHT and 4,6-Di-tert-butyl-m-cresolby fractional distillation. Recrystallization gives the desired purity up to food grade purity (Bayer
AG 2001).
BHT is used as an antioxidant which finds many applications in a wide variety of industries. It is
used in ground vehicle and aviation gasolines; lubricating, turbine, and insulation oils; waxes,
synthetic and natural rubbers, paints, plastics, and elastomers. It protects these materials fromoxidation during prolonged storage. Highly purified grades are suitable for use in foods to retard
oxidation of animal fats, vegetable oils, and oilsoluble vitamins. It is also used in cosmetics andfood packaging materials such as waxed paper, paper board, and polyethylene. It is important indelaying the onset of rancidity of oils and fats in animal feeds, and in preserving the essential
nutrients and pigment-forming compounds of these foods (ACGIH 1986, Bayer AG 2001).
BHT is used as an antioxidant for food, animal feed, petroleum products, synthetic rubbers, plastics,
animal and vegetable oils, and soaps. It serves as an antiskinning agent in paints and inks (Merck
1996).
The above described manifold use pattern of BHT is confirmed by information from European
product registers (queries of 2001 and 2002).
In the following table the worldwide distribution percentages for BHT uses are given. The figures
are related to the year 2000 (Bayer AG, 2002):
Rubber

27 %Plastics

27 %Mineral oil /fuel additive

17 %Foodstuff / pharmaceuticals / cosmetics

12 %Animal feed / pet food

11 %Printing inks / miscellaneous

6%

UNEP PUBLICATIONS

7

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

2.2

Environmental Exposure and Fate

2.2.1

Sources of Environmental Exposure

Releases into the environment may occur during production of BHT as well as during its use indifferent applications as stabilizer and during the use of the products that contain the substance.
Releases during production:
Readily available information on exposure from production of the chemical in the sponsor countryat Bayer AG is stated in the following.
Waste water leaving the production facility is lead into an industrial biological waste water
treatment plant (WWTP). BHT is monitored weekly at changing days at the outlet of the WWTP.
All values from January 2000 to May 2001 are below the determination limit of 2 µg/l. As worst
case for the receiving water a PEC of < 0.003 µg/l is calculated, taking the determination limit of 2
µg/l and the 10 percentile of the receiving river low flow into account. Sewage sludge from theindustrial WWTP is burnt. Thus there is no emission to the geosphere by sludge application (Bayer
AG 2001).
The exhaust from production of BHT is connected to a thermal exhaust purification plant (TAR).
Thus during normal operation no BHT is emitted. Following the last Official Emission Declarationin 2000 less than 25 kg/a BHT were emitted into the atmosphere (Bayer AG 2001).
No data about releases at other production sites are available.
Releases from use of BHT:
Release data from applications of BHT are not readily available.
Release from products containing BHT:
A significant release into the environment is expected from migration of BHT onto the surface of
products containing the substance (BUA report 58, 1991).
Several examinations exist that show the migration of BHT from plastic films into foodstuff andcosmetics. The migration rate depends on the chemical structure of the polymers, the auxiliary
products (e.g. plasticisers) contained therein, the contents and the storage temperature and period(BUA report 58, 1991).
Release from abrasion of tires was estimated for Germany to 40 t/a, assuming an average BHT
content of 0.5 % in the tire tread, 80 000 t/a tire abrasion and a market share of BHT of less than 10% (BUA report 58, 1991).
Monitoring data:
In 1991, a special monitoring program of BHT with a very low determination limit was conductedin German rivers showed the following concentrations (determination limit 0.02 µg/l):
Rhine: < 0.02-0.09 µg/l;

90 percentile 0.08 µg/l

Danube:< 0.02-0.16 µg/l;

90 percentile 0.09 µg/l

Neckar:< 0.02-0.09 µg/l;

90 percentile 0.08 µg/l

(LFU Baden-Württemberg 1994).
8

UNEP PUBLICATIONSOECD SIDS
2.2.2

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Other Information on Environmental Fate

The environmental distribution of BHT was calculated according to the Mackay fugacity modellevel I, considering the measured values of vapor pressure (1.1 Pa), log Kow (5.1) and two values ofwater solubility (0.6 mg/l and 1.1 mg/l). The main target compartment for the compound wasestimated to be air (79.3-87.5 %) followed by soil (6.1-10.2 %) and sediment (5.7-9.5 %) (BayerAG, 2001b+c). The calculated Henry’s law constant in the range of 220 – 404 Pa x m³/mol (BayerAG, 2001a) indicates rapid volatilization from aqueous solution according to the criteria of Thomas(1990). It should be noted, that due to the instability of BHT in aqueous solution, the Mackay
calculation as well as the Henry's law constant give a tendency for BHT distribution among various
environmental compartments and help to identify air as the most important target compartment.
The stability of BHT in the environment and the formation of degradation products depend on
several parameters like presence of atmospheric or dissolved oxygen, irradiation, pH-value,
temperature, presence of traces of heavy metals (metal oxides), moisture, presence of soil and soil
microorganisms (Bayer AG 2001; Mikami et al. 1979a).
A calculation of the indirect photodegradability of BHT in air by hydroxyl radicals according toAtkinson revealed a half-life of 7.0 hours (Bayer AG, 2001d).
Mikami et al. (1979a) studied the photodegradation of 14C-BHT in water. After 8 days of exposure
to natural sun light 98.5 % of applied radioactivity could be recovered from the aqueous solution.
As BHT is not stable under the applied conditions, only 25 % of this sum were unchanged BHT.
BHT-OOH (5.7 %), BHT-OH (4.2 %), BHT-CH2OH (7.5 %), BHT-CHO (2.7 %), BHT-COOH(4.7 %) were determined as degradation products. Polar unidentified products amounted to approx.
48 % of applied radioactivity. A dark control was used to examine the products of thephotochemical process. In the control experiment 59.6 % unchanged BHT, 2.6 % BHT-OOH, 8.8 %BHT-OH, 1.1 % BHT-CH2OH, 3.0 % BHT-CHO, 1.4 % BHT-COOH, and 22.9 % unidentified
polar compounds were observed (see Figure 1) . From the results it can be concluded that BHT is
unstable in water with and without irradiation. Volatilization from the system was very low. The
authors assume that BHT-OOH and BHT-OH were mainly derived from autoxidation of BHT inwater, while oxidation of the methyl group is accelerated by sunlight. Furthermore sunlightirradiation appreciably increased the amounts of the unidentified polar products (see above).
The instability of BHT in aqueous solution was also found by Chang & Maurey (1985). The
reaction of BHT with water and oxygen leads to a large variety of products. The main degradation
product was identified as a stilbenequinone and its precursor, a dimer of BHT.
Inui et al. (1979b) investigated the fate of 14C-phenyl-BHT in an aquatic model ecosystem
consisting of an aquarium which was divided into two compartments. In the larger compartment
soil was spread on the bottom. Three experiments were performed: In test A and C the 14C-phenylBHT was applied to the soil and in test B the substance was added as aqueous emulsion to thesystem. Independent of the mode of application it was shown that BHT is not stable in the test
system; already within the first two days of observation appreciable amounts of degradation
products were formed. In this investigation 14C-phenyl-BHT was mainly decomposed tointermediates like BHT-COOH and BHT-OH. The distribution of radioactivity between thecompartments water, soil and fish was measured at day 7, 14 and 28. In the experiments A and C
between 62 and 74 % of the applied radioactivity remained in soil, 9 – 32 % was dissolved into
water and only a very small part was found in fish. Recovery of 14C in the system was between 85
to 97 % showing that volatilization was only of minor importance. In experiment B about 30 % of
applied radioactivity remained in water, 5 to 14 % was adsorbed to soil and 3 to 10 % wastranslocated in fish. Recovery of total 14C was only 40 to 50 %, suggesting that more than 50 % was
evaporated into atmosphere.
UNEP PUBLICATIONS

9OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

The stability of BHT in soil was determined with three different soil types (light clay, sandy clay
loam, sandy loam) under sterilized and non-sterilized conditions. According to the results obtained,
BHT is relatively unstable in the three soils tested: With non sterilized soils about 63-82 % of BHT
were decomposed after one day (about
1-2 % mineralized to CO2) and 77-92 % (21-29 %mineralized) after 24 days of incubation. Under sterilized conditions 25-35 % BHT were
decomposed after one day and 27-41 % after 24 days, mineralization was negligible (< 2 %). After
one day
57-68 % of BHT and after 24 days 50-61 % remained unchanged. Under non-sterilized
conditions the amount of total volatile 14C was 26 to 42 % (21 to 29 % 14CO2) Under sterilized
conditions the amount of volatile 14C was 43 to 56 % after 24 days (< 2 % 14CO2). From theseresults it can be concluded that BHT is altered to nonvolatile products mainly by biologicalprocesses. In soil more than 10 degradation products were found. As major decomposition products
BHT-OOH and BHT-OH were identified (Mikami et al. 1979b). The following possible
degradation pathway for BHT in soil was proposed by the authors (Figure 1):

Depending on the exposure pathways, the compartments air, hydrosphere and soil can beenvironmental target compartments for this substance and its metabolites.
From the available experimental data BHT is not expected to be readily biodegradable in surface
waters as well as in sewage treatment plants. In a modified MITI test according to OECD guideline
301 C, an unadapted mixed microbial inoculum mineralized 4.5 % BHT within 28 days of
incubation (MITI 1992).
The bioaccumulation of BHT in fish (Cyprinus carpio) was determined in a test according toOECD guideline 305 C. Bioconcentration factors (BCF) in the range of 230 - 2500 after 56 days
were reported (MITI 1992). No explanation of the high variation of results is given and only ageneral test procedure without mentioning analytical details is reported. Therefore it can not be
decided whether the proven instability of BHT in aqueous solution and in the presence of oxygen
was adequately taken into account in these tests. In contrast to the high BCFs determined by MITI
(1992), Inui et al. (1979) found lower values for fish (carp), snails, daphnids and algae in a model
ecosystem. The BCF values determined after a 28 days exposure period were 2-17 for fish, 30 forsnails and 38 for algae. However, this study exhibits methodological deficiencies concerning
number of exposed test organism and analytical test procedure. Low BCF values were also reportedby Geyer et al. (1986), who reviewed data published on the concentration of BHT in human fat
tissue. BCFs calculated from BHT concentrations in fat tissue and mean BHT uptake from food

10

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

were in the range of 0.30 – 0.98 indicating no substantial risk for accumulation of BHT from food.
However, these BCF values cannot be compared with BCF values determined for aquatic species.
From the wide range of available BCF values, it can be assumed that BHT has a moderate to highbioaccumulation potential in aquatic species.
2.3

Human Exposure

2.3.1

Occupational Exposure

Exposure of workers to BHT may occur during production, processing and use of the chemical,
with the dermal and inhalation routes being the principal routes of exposure.
To protect workers several precautionary and protective measures are taken. These measures
include technical equipment, e.g. the use of automatic filling machines and suction devices at fillingand sampling stations to minimize exposure to dust. The measures also include appropriate personal
protection equipment which is prescribed in detail for different work situations, such as sampling,
maintenance, and repair work. For sampling, devices without dead volume are used, and thepersons involved have to wear goggles and gloves. Depending on the work to be done during
maintenance, gas filter masks or a respirator with independent air supply have to be used as well asfull protective clothing.
A workplace limit concentration of 10 mg/m³ is laid down in several countries including Germany.
The German occupational exposure limit (OEL) value is laid down in TRGS 900 with 10 mg/m³,
this value is originating from the Netherlands in 1997.
Information on workplace exposure during production of BHT at Bayer AG/Germany is available.
Already in 1985 all stations at the production facility, where exposure to BHT could have occurred,
had been monitored in a precautionary program. All measured data were below ¼ of the today
effective OEL value of 10 mg/m³, except at the filling station with the highest value of 2.7 mg/m³.
As a consequence of the measurements at the filling station, the filling station was automated. Since
all other measured values were below ¼ of the OEL value as of April 1997, and no changes in the
production facility had taken place, no further measurements were performed in agreement with theguidance document TRGS 402 (Bayer AG 2001).
It is not known whether or not the same risk management techniques are used in processing plants.
2.3.2

Consumer Exposure

BHT is a registered antioxidant (EG Antioxidant Directive E321), licensed for food products,
animal feed, cosmetics, and packaging material. Taste, odor, and color alterations of fatty foodcomponents (especially of animal origin which do not have natural antioxidants like vegetable fats)
as well as oxidative spoilage of vitamin A, K and carotinoids are slowed down during storage by
the antioxidant, when the nutrients are exposed to heat, light, and metal traces.
For the different regions of the world there are directives regulating the use and the allowed amountof BHT in food products, animal feed and packaging material. Dietary intake is the main source ofconsumer exposure. Other exposures via the environment are considered to be comparatively low.
The acceptable daily intake (ADI value) of BHT by WHO/FAO is given with maximal 0 - 0.3
mg/kg bw/day. In a worldwide evaluation about consumption of BHT by food, the Joint FAO/WHO
Expert Committee on Food Additives (JECFA) concluded that the ADI for BHT is unlikely to be
exceeded on the basis of the estimated intakes in the 10 countries for which data were available(WHO 1996).
UNEP PUBLICATIONS

11

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

3

HUMAN HEALTH HAZARDS

3.1

Effects on Human Health

3.1.1

Toxicokinetics, Metabolism and Distribution

BHT is readily absorbed through the gastrointestinal tract and, to a small extent, through the intact
skin. After long-term feeding of BHT-containing diets the compound is accumulated especially inadipose tissue, while lower levels are found in the liver, with elimination half-lives of 7 - 10 days
for both organs on cessation of treatment. In rats and probably also in humans, an enterohepatic
circulation takes place, particularly for the metabolite BHT acid and its glucuronide. BHT is
excreted primarily in the urine and, to a smaller extent, in the faeces. After a single oral application
to rats 80 - 90% of the dose was found in the urine within four days, most of it within 24 hours.
Rabbits excreted approximately 54% within four days and humans 66% within 11 days in urine.
Several metabolic pathways and metabolites have been identified. BHT is activated by acytochrome P450 dependent metabolic reaction in the liver (BUA 1991).
3.1.2

Acute Toxicity

Inhalation
No data available
DermalIn a test according to OECD Test Guideline 402 (Hazleton France 1988b), the LD50 was above thesingle dermal dose of 2000 mg/kg bw BHT administered to Sprague-Dawley rats as an aqueous
dispersion in gum arabicum (10% w/v). No effects were observed with regard to clinical signs,
body weight and gross examination.
Conclusion
BHT is of low acute toxicity following dermal application. The LD50 after acute dermal exposure
was > 2000 mg/kg bw.
Oral
In an acute oral toxicity test with Sprague-Dawley rats conducted according to OECD Test
Guideline 401 (Hazleton France 1988a), up to 2930 mg/kg bw BHT were administered as anaqueous dispersion in gum arabicum (10% w/v). No effects were observed for clinical signs, bodyweight and gross examination. Likewise, no effects were noted in male and female rats after single
application of 10000 mg/kg bw BHT as suspension in propylene glycol (Spanjers & Til 1978;
Bomhard 1996). Some earlier, not well documented studies described lower LD50 values (e.g. 1970
mg/kg bw purified commercial product; vehicle: corn oil (Deichmann et al., 1955)), that could not
be verified by the adequately performed guideline study (Hazleton France 1988a). The study
conducted in accordance with OECD test guidelines is considered more appropriate for thisendpoint.
Two cases of acute intoxication were reported in which two adult women (22 and 24 years old)inadvertently ingested BHT (4 g and 80 g) on an empty stomach. After treatment of the symptoms(e.g. severe epigastric cramping, nausea, vomiting, neurological disorders) complete recovery
occurred within a few days (Grogan 1986; Shlian & Goldstone 1986).
Conclusion
12

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

BHT is of low acute toxicity following oral administration. The LD50 after acute oral exposure was> 2930 mg/kg bw.
3.1.3

Irritation

Skin Irritation
After 24-hour semi-occlusive application of BHT (500 mg undiluted) to the intact and scarified
skin, respectively, a very slight irritation could be noted. On the intact skin, erythemas occurred in
3/6 and 2/6 animals after 24 hours and 72 hours, respectively; edemas occurred in 1/6 animals after
24 hours and none after 72 hours. On the abraded skin, erythemas and edemas occurred in 1/6
animals after 24 hours and none after 72 hours (Bomhard 1996).
Conclusion
BHT was slightly irritating to the skin of rabbits.
Eye Irritation
When tested in the Draize test (Bomhard 1996), 6 of 6 rabbits showed slight conjunctivitis 24 hours
after application of 100 mg BHT (undiluted). Symptoms were completely reversible after 72 hours.
Conclusion
BHT was slightly irritating to the eye of rabbits.
3.1.4

Sensitisation

Studies in Animals
There are no relevant experimental data available. Limited studies with guinea pigs showed no
indications of a sensitizing potential (Bayer AG 2001).
Studies in Humans
Despite of being in wide dispersive use for years, only a few cases of skin sensitization due to BHT
as ingredient of various products are discussed. Signs of contact dermatitis after dermal exposure to
BHT and allergic reactions after oral intake of a mixture of BHT and butylated hydroxyanisole(BHA) were occasionally reported (DFG 1985; Flyvholm & Menne 1990). In another report(Goodman et al. 1990), two patients with chronic idiopathic urticaria developed exacerbations whenchallenged with BHT/BHA, but had less symptoms after consuming a BHT/BHA-free diet.
When patch tested on more than 15 individuals, BHT showed mild skin irritation; a positive skinreaction 14 days later was interpreted as sensitization (Mallette & von Haam 1952). However, theselimited reports do not allow drawing any conclusions as to the skin irritation and sensitization of
BHT in view of the widespread exposure to BHT in consumer products. There are some more
recent patch test results obtained from the medical surveillance of great numbers of workers (deBoer et al. 1989; Goh & Ho 1993) or patients (Kanerva et al. 1997; 1999) which were all negative.
However, sensitization reactions cannot be fully excluded in single cases.

UNEP PUBLICATIONS

13OECD SIDS
3.1.5

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Repeated Dose Toxicity

Sub-acute and sub-chronic effects
Sub-acute exposure to BHT resulted in haemorrhagic effects and, at high doses, subsequently indeath. In a 40 day feeding study with male Sprague-Dawley rats (ca. 436-874 mg/kg bw/day)(Takahashi & Hiraga 1978a), doses from approximately 500 mg/kg bw (0.69% in the diet) onwards
were fatal, with massive haemorrhages occurring in the pleural and peritoneal cavities. Surviving
animals also showed signs of haemorrhage in other organs such as epididymis, testis and pancreas.
A dose-dependent decrease in the prothrombin index (15-35%) was found in the survivors of alldose groups.
In a combined species study, species, strain and sex differences were found. Haemorrhagic deaths
were noted in male rats of strains Sprague-Dawley, Wistar, Donryu and Fischer and in female
Fischer rats (1.2% BHT in diet corresponding to between 638-1120 (m) and 854-1000 (f) mg/kg
bw/day depending on strain; 3 weeks). Six different mice strains (only males) were negative in thisrespect (847-1925 mg/kg bw/day, 1 week), with the exception of haemorrhagic deaths occurring in
ddY strain. No such effects were seen in dogs (173, 440 or 760 mg/kg bw/day given in diet, 2
weeks), hamsters (380 or 760 mg/kg bw/day i.p., 3 days), guinea pigs (190 or 380 mg/kg bw/day
i.p., 3 days), rabbits (177, 242 or 390 mg/kg bw/day in diet, 2 weeks) and quails (1% in diet = ca.
1056 mg/kg bw/day, 17 days) (Takahashi et al. 1980).
The effect on the prothrombin index (PI) observed by Takahashi & Hiraga (1978a) was also foundto be species-specific and confined to rats and mice. Significant decreases of this index were notedin the above study at all dose levels in all rat strains (18 - 92% of control) and, except for ICR mice,in all mouse strains, although mice reacted much less markedly (PI 79 - 96%) than rats (Takahashi
et al. 1980). However, the number of animals used in these studies was limited and the doses werereportedly not exact. In a re-examination study with larger numbers of animals and higher
exposures, oral administration of BHT resulted in lung haemorrhages in all dose groups of mice,but only when housed in mesh-bottomed cages (660-2860 mg/kg bw/day; 21 days), not in cageswith soft-wood chips (1570-5470 mg/kg bw/day; 30 days). A protective effect of cedar oil
contained in bedding chips is discussed. PI was significantly reduced in most dose groups (up to
60% of control). No haemorrhages, but PI reduction of up to 77% of controls occurred in guinea
pigs (Takahashi 1992).
At lower doses, the PI decrease was found to be transient in Sprague-Dawley rats (Takahashi &Hiraga 1978b): after administration of doses as low as 0.017 % (ca. 15 mg/kg bw/day) in the dietfor one week, a significant effect was observed, while at four weeks only the highest dose group(0.5 % in the diet; ca. 326 mg/kg bw/day) showed a decrease in this index. It should be noted thatthe decline in prothrombin indices measured after 1 week (100, 90.5, 85.9, 82.2, 78.4, 80.9, 84.4
and 73.8% at 0, 7.54, 14.7, 34.1, 62.5, 129, 227 and 529 mg/kg bw/day, respectively) is notmarkedly dose-related and only five animals were used per group.
The major causative factor for the mechanism underlying the pathogenesis of haemorrhages ispossibly the BHT-related decrease of vitamin K-dependent blood coagulation factors as studied inexperiments with Sprague-Dawley rats (Takahashi & Hiraga 1979; Takahashi 1986; 1988). Rats
given 1.2% BHT in the diet for 1, 2, 3, 4, 5, 6, or 7 days (average intake about 1000 mg/kg bw/day)showed a significant reduction in plasma concentration of factors II, VII, IX and X in a timedependent fashion (Takahashi 1986). Indirect evidence of this antagonistic effect of BHT on
vitamin K was found in 3-week oral studies, in which the simultaneous administration ofphylloquinone (vitamin K1) had a preventing effect on haemorrhages and PI reduction (Takahashi& Hiraga 1979). BHT quinone methide is believed to be the active metabolite affecting the vitamin
K redox cycle (Takahashi 1988).
14

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Other effects caused by subacute (6 - 26 days) doses of BHT after oral administration to rats include
liver dysfunction and histopathologic changes (> 25 mg/kg bw/day, 7 days; liver enlargement,
centrilobular necrosis, glutathion depletion, increase in transaminases, enzyme induction, increases
in phospholipids and cholesterol), effects on the kidney and thyroid gland (DFG 1985).
Lung damage was noted in a sub-chronic study (Miyakawa et al. 1986) in which CD-1 mice weredermally treated with BHT (males: 145-867 mg/kg bw/day; females: 208-1245 mg/kg bw/day) for
four weeks. Between day 4 and 8 of the study dose-dependent respiratory distress with subsequentmortality was observed in all dose groups, except for males at 145 mg/kg bw/day. Autopsy revealed
congestion and enlargement of the lung; histologically, degeneration and necrosis of the type I
alveolar epithelial cells and an increased number of type II cells was found. The skin at the site ofapplication showed epidermal hyperplasia. Other organs appeared normal in all dose groups.
Similar treatment of F 344 rats (ca. 2000 mg/kg bw/day) and of hamsters (ca. 3100 mg/kg bw/day)
was tolerated without adverse effects other than a slight growth retardation.
In a 28-days oral study, Rhesus monkeys (Macaca mulatta) received daily doses of (i) 500 mg/kg
bw (infants; 3 animals), (ii) 500 mg/kg bw (juveniles; 3 animals), (iii) 50 mg/kg bw (juveniles; 2animals) by intragastric intubation (Allen & Engblom 1972). No mortalities or clinicalabnormalities were observed. Haematology, urinalysis and blood biochemistry determined by week
2 and 4 were unaffected. There was no effect on relative liver weight. Slightly modified enzyme
activity of hepatic microsomes were found in 500 mg/kg juveniles group (statistically significant
increase of nitroanisole demethylase activity by week 2 and 4 and decrease of glucose-6phosphatase activity by week 4), but not in 50 mg/kg juveniles or 500 mg/kg infants group. There
were no effects on protein content, total lipids, RNA, DNA or cytochrome P450 in the liver of alltreated groups as compared to controls. Histopathological evaluation showed an indication ofhepatocytomegaly and an enlargement of hepatic cell nucleoli in the juvenile animals. In the 500
mg/kg groups of infants and juveniles, moderate proliferation of hepatic endoplasmic reticulum,
nucleolar fragmentation and presence of large intranuclear fibrils was observed in many of thehepatic nuclei. The 500 mg/kg group of infants was much less responsive. The 50 mg/kg group ofjuveniles showed less obvious cytoplasmic changes and no nucleolar changes. This lower responseof infants as compared to juveniles is probably related to the lower activity of drug-metabolizing
enzymes and is strongly suggesting that metabolites were responsible for most of the changes
observed. Histopathological evaluation of all organs other than liver from either infant or juvenile
animals showed no major BHT-related pathological changes.
A 28-day oral study with Wistar rats (Powell et al. 1986) can be considered as critical study forderiving a NOAEL. The study focused primarily on hepatotoxic effects since these seem to play acausative role in the development of BHT-related liver tumours (see chapter 3.10). BHT was given
to groups of male Wistar rats by gavage at doses of 0, 25, 250 and 500 mg/kg bw/day for 7 and for
28 days, respectively. There were no significant BHT-related effects on the body weight. At thehighest dose level, progressive periportal hepatocyte necrosis was observed after 7 and after 28 daysof treatment. These lesions were associated with bile duct proliferation, fibrosis, hepatocyte
hyperplasia and hepatocellular hypertrophy. At 250 mg/kg bw/day only slight evidence of cell
damage as indicated by glycogen accumulation was seen after 7 and after 28 days. Dose-related
effects were observed in the mid- and high-dose groups with regard to hepatomegaly andbiochemical parameters for both time points (microsomal activity of ethoxycoumarin-O-deethylase
and epoxide hydrolase). Dose-dependent increase was seen for hepatic microsomal protein content
after 28 days of exposure while hepatic glucose-6-phosphatase activity decreased with increasing
dose and exposure. 25 mg/kg bw/day is considered as no-observed adverse effect level (NOAEL)
for both 7 and 28 days of treatment.

UNEP PUBLICATIONS

15

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Chronic effects
In a long-term feeding study (Williams et al. 1990a) which was not designed as typical chronic
study, but focused on the possible development of hepatocellular foci, male Fischer 344 rats were
fed diets containing 100, 300, 1000, 3000 and 6000 ppm BHT, which was equivalent to about 7.5,
23, 75, 225 and 450 mg/kg bw/day. After random selection, part of the animals was sacrificed at 12,
36 and 48 weeks and examined, while the remaining animals were exposed for 76 weeks. In the twohighest dose groups, body weight gain was reduced. Only rats fed 6000 ppm BHT revealed asignificantly increased liver weight. After the different treatment periods the number ofphenotypically altered hepatic foci (AHF) was not significantly different compared with respective
controls. Similar findings were obtained in a second study (Williams et al. 1990a), in which rats
received diets containing 0 and 12000 ppm BHT (ca. 900 mg/kg bw/day) for 110 weeks, though theincidence of AHF was slightly decreased. With regard to the examined parameters, i.e. body andliver weight and histopathology, the NOAEL derived from the first study was 1000 ppm BHT (ca.
75 mg/kg bw/day). Results on carcinogenic effects are summarized in chapter 3.10.
In a two-generation carcinogenicity study (Olsen et al. 1986), BHT was administered in the diet tomale and female Wistar rats at doses of 0, 25, 100 or 500 mg/kg bw/day (F0 generation) until
mating (week 13) and, in the case of female rats, until the end of the lactation period. Groups of theF1 generation received the above doses until the age of 141 - 144 weeks, with the exception of 250
instead of 500 mg/kg bw/day because of nephrotoxic effects in F0 female rats in the highest dosegroup. In this dose group, also body weight gain of the F0 animals was significantly reduced ascompared to controls despite of unaffected food consumption. The same effect was observed in the
BHT-treated animals of the F1 generation, though in all exposed groups and dose-related. Survival
rates increased with increasing doses. Blood analysis (no data given) and serum chemistry was only
done in the high dose group (F1) and were normal, with the exception of reduced levels of serum
triglycerides in both sexes. The lowest dose used in this study, i.e. 25 mg/kg bw/day, can beconsidered as the NOAEL. The results on neoplastic lesions are summarized in chapter 3.10.
Another two-generation feeding study with Wistar rats conducted by the Robens Institute (Price
1994; McFarlane et al. 1997) focused primarily on hepatocellular effects in the F1 generation inorder to elucidate the findings of the Olsen et al. (1986) study with respect to hepatocellular
carcinomas (see chapter 3.10). However, since also effects on kidney, thyroid and adrenals wereassayed, the NOAEL derived from this study refers to all relevant possible target organs. Strain(Wistar) and dosing regime (0, 25, 100 or 500 mg/kg bw/day BHT (F0); 0, 25, 100 or 250 mg/kg
bw/day BHT (F1)) were similar to those in the study conducted by Olsen et al. (1986). At weaning,part of the male pups was selected for the respective examinations, while the remaining male F1 rats
received BHT containing diet until the time points 4 weeks, 6, 11, 16 and 22 months after weaning.
Dams of the F0 generation showed increased relative liver weight and enlarged and eosinophilic
hepatocytes in the high-dose group (500 mg/kg bw/day).
The significant signs and symptoms found in the F1 progeny are as follows:

16•

Body weight: reduced only in mid- and high-dose group (100 and 250 mg/kg bw/day BHT)
throughout 22-month treatment period.

•

Pathology and histopathology of liver: significant effects only in high-dose group, i.e.
increase of (i) relative liver weight (until 16 months after weaning); (ii) enlarged andeosinophilic centrilobular hepatocytes (from 6 months onwards); (iii) periportal induction of
gamma-glutamyl transferase (from 11 months); (iv) altered hepatic nodules (at 16 months)(Small eosinophilic altered hepatic foci (AHF) and nodules between 11 - 22 months present
in all dose groups and controls)

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)•

Immunocytochemistry for AHF immunostain types cytochrome P450 (1A and 1B), epoxidehydrolase (only performed with high-dose group):
Increase of (i) AHF 1B type (16 months), (ii) total AHF (11, 16, 22 months)•

Parameters of xenobiotic metabolism (liver biochemistry): Increase of (i) total cytochrome
P450 (high-dose group at 11 and 16 months); (ii) epoxide hydrolase, glutathione-Stransferase and pentoxyresorufin-O-depentylase (dose-related; starting at 21 days of age;significant in mid- and high-dose groups); (iii) ethoxyresorufin-O-deethylase (but notstatistically significant).

•

Histopathology of kidneys: no indication of exacerbation of chronic progressive
nephropathy (CPN) that was observed in all rats (incl. controls) from 11 months, but witheven slightly less severity in the high-dose group. No adverse effect of BHT on the adrenals
at time points examined (11, 16 and 22 months).

•

Microscopic examination of thyroid (presumably only at 11, 16 and 22 months): marked
thyroid hyperactivity only in mid- and high-dose group from 11 months; less severe changes
in mid-dose group.

•

Total serum thyroxin: not affected at any dose level measured at 16 and 22 months.

Based upon the described effects in liver, kidney, thyroid and adrenals, 25 mg/kg bw/day BHT canbe considered as NOAEL.
In a 104-week feeding study (1 and 2% BHT in diet; 1640/3480 (males) and 1750/4130 mg/kg
bw/day (females)) with B6C3F1 mice (Inai et al., 1988), dose-dependent reduction of body weightgain was noted. In males, dose-dependent nuclear pleomorphism of hepatocyctes occurred andabsolute and relative liver weights were increased. Survival of males was increased. 2% in the dietwas considered as MTD. However, due to the assumed inhomogeneous distribution of BHT in thefeed pellets the doses given do not seem valid.
Conclusion
Long-term exposure to BHT can result in functional and histological changes of lung, liver, kidneys
and thyroid. Higher sub-acute and sub-chronic doses of BHT can cause death of mice or rats, either
due to severe lung damage or massive haemorrhages. In the case of chronic oral exposure, liver andthyroid are the main targets. Doses above 25 mg BHT/kg bw/day result in thyroid hyperactivity,
enlargement of the liver, induction of several liver enzymes. These hepatic effects, together with theformation of preneoplastic foci and nodules are discussed as causative factors in the mechanism of
non-genotoxic carcinogenicity of BHT (see chapter 3.10). As derived from rat studies, a dose of 25
mg BHT/kg bw/day can be considered as reliable NOAEL for chronic exposure.The haemorrhagic
effects of high repeated doses of BHT seen in certain strains of mice and rats, but not in otherspecies, may be related to its ability to interact with prothrombin and vitamin K.
3.1.6

Mutagenicity

In vitro Studies
BHT did not cause gene mutations in the Ames test with and without metabolic activation using thestandard test strains S. typhimurium TA98, TA100, TA1535, TA1537, TA1538. No data were
given on cytotoxicity, but the highest concentration tested was 10000 µg/plate (Williams et al.
1990b). A bacterial gene mutation assay with S. typhimurium strains TA102 and TA2638, E. coli
WP2/pKM101 and WP2 uvrA/pKM101 with metabolic activation was also negative (Watanabe et
UNEP PUBLICATIONS

17

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

al. 1998). As reviewed by Bomhard et al. (1992), various Ames tests with and without metabolic
activation were negative as were two other bacterial gene mutation tests with E. coli strains.
A HPRT assay with adult rat liver cells (ARL line 18) was negative at concentrations of 50, 60, 70,
80, 90 µg/ml (Williams et al. 1990b). Another HPRT assay with Chinese hamster V79 cells was
positive only at cytotoxic concentrations (Paschin & Bahitova 1984). A mouse lymphoma assay
was reported as positive with metabolic activation and inconclusive without metabolic activation.
However, small and large colonies were not distinguished in this test and the results of three
independent trials are contradictory concerning effects; non-cytotoxic concentrations did not induce
an increase in mutation frequency (with and without S9-mix) (McGregor et al. 1988).
Conclusion
BHT showed no potential to cause point mutations in bacterial and mammalian test systems.
A chromosome aberration assay with CHO cells (concentrations tested: 1.6 - 16 µg/ml) was
negative without metabolic activation. With metabolic activation a slight increase and positive trend
was observed, but the authors considered the overall result as negative (Galloway et al. 1987). In atest conducted by Grillo & Dulout (1995) without metabolic activation, BHT (0.1 - 0.5 µg/ml)
induced a significant increase of chromatid-type aberrations. As reviewed by Bomhard et al. (1992),
other chromosomal aberrations tests were negative (2 with CHL cells; 1 with V79 cells); a test withhuman embryonic lung cells (cell line WI-38) was positive, but the test system (analysis ofanaphases) is not validated (Newell & Maxwell 1972; Maxwell & Newell 1974)
In the sister chromatid exchange (SCE) assay, BHT (0.1 - 0.5 µg/ml) was negative in both CHO
cells and human lymphocytes (Grillo & Dulout 1995). As reviewed by Bomhard et al. (1992), sixother SCE tests (4 with CHO-, 1 with CHL-cells, 1 with DON hamster cells) were also negative.
Negative results were also reported in a DNA damage and repair assay (UDS test) using primary rat
hepatocytes in the concentration range of 0.01 - 10.0 µg/ml (Williams et al. 1990b).
BHT did not induce DNA repair in the umu-test with Salmonella typhimurium TA1535/pSK 1002
in the concentration range of 0.4 - 6 mM (no cytotoxicity) (Heil et al. 1996).
Conclusion
Overall, the available studies demonstrate that BHT has no overt clastogenic activity in vitro.
In vivo Studies
A mouse specific locus test involving lifetime exposure of males to ca. 1000 mg/kg bw per day withthe diet was negative (Cumming et al. 1976).
A bone marrow micronucleus assay with mice (i.p. injections of 0, 125, 250, 500 or 1000 mg/kg bw
per day, 5 days; only females) was negative. There are no concrete informations given concerning
toxic effects of dosing but the highest dose applied was within a factor of 2 of the approximate LD50
established first (Bruce & Heddle 1979). In another micronucleus assay with mice (Paschin et al.
1986) only one single dose level was applied (i.p. injection of 75 mg/kg bw), but the negative result
is considered as supportive for the other test result.
There are several chromosome aberration tests available which showed no clastogenic effects onbone marrow chromosomes of rats or mice:•

18

after administration of 1.5% BHT in the diet (ca. 750 mg/kg bw per day) to Wistar orSprague-Dawley rats for 9 months (Tokyo Metropolitan Research Laboratory of Public
Health 1978) (Limitations: Only one dose; no data on positive control)
UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)•

after five oral administrations of BHT (30, 250 or 500 mg/kg bw/day) to male random bredalbino rats (Newell & Maxwell 1972; Maxwell & Newell 1974) (Limitation: metaphases
investigated once) after single oral administration of BHT (30, 900 or 1400 mg/kg bw) tomale random bred albino rats (Newell & Maxwell 1972; Maxwell & Newell 1974)•

after administration of 1.5% BHT in the diet (ca. 2000 mg/kg bw per day; based on standard
diet conversion factor) to ICR mice for 9 months (Tokyo Metropolitan Research Laboratoryof Public Health 1978) (Limitations: Only one dose; no data on positive control).

A positive result was reported for a dominant lethal assay with rats fed with 0.04, 0.13 or 0.4%
BHT in the diet (50, 166 or 500 mg/kg bw/day) for 10 weeks (Sheu et al. 1986). However, recalculation of the dead implants according to recent guidelines for the method of calculating rates ofdominant lethal mutations (Dean 1983; EEC 1987; OECD 1984) revealed no differences between
BHT-treated animals and controls. In a parallel study with mice receiving 1 % (w/w) BHT in thediet (ca. 1500 mg/kg bw/day) for 8 weeks, the dominant lethal assay was negative (Sheu et al.
1986). As reviewed by Bomhard et al. (1992), two other dominant lethal tests with rats and two
with mice were also negative.
Furthermore, the heritable translocation assay with mice (1% BHT in diet, 8 weeks) was negative(Sheu et al., 1986).
The above clastogenicity and chromosomal aberration studies were not conducted in accordancewith specific testing guidelines. Most of these studies have limitations as noted above, but taken
together they allow drawing conclusions as to the clastogenic potential of the substance.
Conclusion
BHT showed no potential to cause point mutations in in vivo test systems. Overall, the available
cytogenicity studies demonstrate that BHT has no clastogenic activity in vivo.
3.1.7

Carcinogenicity

Carcinogenic effects
In several reviews (e.g. IARC 1986, WHO 1996), limited evidence for carcinogenicity of BHT forexperimental animals was found and this was based on several oral studies with mice and rats.
Either no differences in tumour incidences among exposed and control animals were found orinconsistent, i.e. increased incidences at low, but not at high doses, were noted. Of the more recent
studies, two reports indicate an increased rate of hepatocellular tumours in mice (Inai et al. 1988)
and rats (Olsen et al. 1986). Prompted by these results, three other studies (Price 1994; two by
Williams et al. 1990a) were conducted with rats, which however could not verify these results.
These five chronic studies have been summarized in chapter 3.8. The aspects concerning
carcinogenicity are discussed as follows.
In the 104-weeks study with B6C3F1 mice (Inai et al. 1988), tumour rates were not different among
exposed and control females. Males showed an increased incidence of liver tumours in all dosegroups including controls, i.e. 38%, 62% and 66% in 0, 1750 and 4130 mg/kg bw groups,
respectively. A statistically significant difference as compared to controls was only noted in thehigh-dose group and only for the incidence of multiple adenomas. Rates of carcinomas did not
differ from controls. Limitations of this study are the rather high doses used, although their quantity
is somewhat unclear due to the assumed inhomogeneous distribution of BHT in the feed, the long
duration of exposure and an increased survival in males with increasing dose. The liver tumour
incidence in control animals was elevated in comparison to earlier studies performed in the samelaboratory as well as compared to historical control data published by NTP (see page 54 of
UNEP PUBLICATIONS

19

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

publication). In addition, the mouse strain used is known to have a relatively high rate of
spontaneous hepatocellular tumours, especially in males, and a high variance of occurrence indifferent studies (Bomhard et al. 1992).
BHT was administered in the diet at levels of 3000 or 6000 ppm (ca. 450 or 900 mg/kg bw per day)to male and female B6C3F1 mice for 107-108 weeks (NCI 1979). The incidence ofalveolar/bronchiolar carcinomas or adenomas was significantly increased in the female mice in the
low dose group, but the incidences were not significantly dose-related (control: 1/20, low dose:
16/46, high dose: 7/50). Thus, these lung tumours in the females cannot clearly be related to theadministration of BHT. In addition, such tumours were not found in other long term studies andtherefore are regarded to be by chance. The incidence of tumours of the liver in male mice and in
the incidence of sarcomas of multiple organs in female mice was significantly reduced comparedwith the control animals.
In a parallel study with male and female Fischer 344 rats (NCI 1979), BHT was administered in thediet at levels of 3000 or 6000 ppm (ca. 225 or 450 mg/kg bw per day) for 105 weeks. No
significantly higher incidences of tumours were found in either male or female rats as compared tothe controls.
In an oral 104-weeks study (Hirose et al. 1981), Wistar rats received 0.25 or 1% BHT with the diet.
Histopathological examinations revealed a variety of tumours at the end of the study; however,there was no dose-response in either type of tumour or total number as compared to controls.
In the two-generation carcinogenicity study conducted by Olsen et al. (1986), dose-related increases
in hepatocellular carcinomas (1%, 0%, 1%, 8% at 0, 25, 100, 250 mg/kg bw) and hepatocellularadenomas (1%, 1%, 6%, 18%) in male Wistar rats exposed in utero, during lactation and thereafter
up to 144 weeks (overall test for heterogenicity and trend) were reported. Increased tumour rates infemale rats were only statistically significant for hepatocellular adenomas when tested for trend. In
both sexes, 25 mg/kg bw/day had no effect. Most tumours were detected in the animals examined at
terminal sacrifice at 141 - 144 weeks, an exposure time that exceeds the duration of conventional
studies. Body weight gain was reduced and survival was increased with increasing doses.
In the 76- and 110-weeks feeding studies conducted by Williams et al. (1990a), no hepatocellular
carcinomas were detected in the male Fischer 344 rats exposed to up to 900 mg/kg bw/day andthere was no BHT-related trend in increase in hepatocellular adenomas, which were detected in all
dose groups including controls.
No tumours were found in the two-generation carcinogenicity study conducted by the Robens
Institute (Price 1994; McFarlane et al. 1997) under test conditions that were almost identical tothose in the Olsen et al. (1986) study, with the following exceptions: (i) only male F1 Wistar rats
exposed to BHT; (ii) exposure up to conventional period of 22 months (104 weeks). The slightly
higher rates of altered hepatic foci and nodules in the high-dose group (250 mg/kg bw/day) may beconsidered as markers for a possible late tumour development if the study would have been
extended. In this respect, also the observed dose-related induction of liver enzymes involved inxenobiotic metabolism (see chapter 3.8) may be of relevance.
In evaluating the above studies BHT was not clearly carcinogenic. An exception is the study
conducted by Olsen et al. (1986). It has been discussed (Bomhard et al. 1992; WHO 1996; Williams
et al. 1999) whether the specific test conditions of this study and not BHT itself resulted in theincreased tumour rates. This has been mainly based on the results of the other two-generation study(Price 1994). Taken together, the following two factors are considered as causative non-genotoxic
factors:

20

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)•

Liver enlargement accompanied by induction of mixed function oxidase enzyme: there isexperience from other studies, e.g. with phenobarbitone, that lifetime duration of such anadaptive response can result in the development of tumours as a "late" phenomenon (Price1994).

•

Malnutrition: In both two-generation studies, body weight gain was significantly reduced in
the progeny of the high-dose parental generation throughout the entire exposure periods. It
has been discussed, but not examined, that a deficiency of the phospholipid phosphatidylcholine resulted from malnourishment during the lactation period. Since choline was notexogeneously supplied with the feed, a long-term choline deficiency finally may havecaused an increase in liver cancer as known from the literature (Price 1994).

It is probable that both factors, alone or in combination, were responsible for the formation oftumours in the study conducted by Olsen et al. (1986). In any case, both explanations imply that atumorigenic response should be expected to occur only after a relatively long exposure period. As amatter of fact, most tumours were detected in the animals examined at terminal sacrifice at 141 144 weeks, an exposure time that exceeds the duration of conventional studies by about 40 weeks.
However, no tumours were observed in the Price (1994) study that terminated after 22 months.
In the Netherlands Cohort Study (Botterweck et al. 2000) in which more than 120000 men andwomen aged 55 - 69 years were followed-up for 6.3 years, no significant association was foundbetween stomach cancer risk and mean BHT (351 µg/day) and butylated hydroxyanisole (BHA)
intake (105 µg/day) through the diet. For comparison, the acceptable daily intake (ADI) establishedby the FAO/WHO Joint Expert Committee on Food Additives is 0 - 0.3 mg/kg bw/day for BHT(WHO 1996).bw/day
Tumour-promoting and anticarcinogenic effects
In several studies, indications of a tumour-promoting activity of BHT were found when given after
an initiating carcinogen for mouse lung and colon, and rat liver and urinary bladder. Blocking ofintercellular molecular transfer at high BHT doses has been assumed as mechanism behind thiseffect. On the other hand, BHT can also exert an anticarcinogenic activity. In several studies, BHT
inhibited the carcinogenesis of potent carcinogens when given to mice or rats at high doses, i.e.
greater than 3000 ppm. For example, BHT inhibited the hepatocarcinogenicity of aflatoxin B1 and
2-acetylaminofluorene in rats. At low doses (100 - 1000 ppm) also anticarcinogenic effects of BHT
were observed probably due to free radical trapping. Whether BHT acts as tumour promotor or as
tumour inhibitor probably depends on the application regime. Application of BHT before or
together with the carcinogen seems to be protective, while subsequent application results in tumourpromoting effects (BUA 1991; IARC 1986; WHO 1996; Williams et al. 1999).
Conclusion
BHT is not a genotoxic carcinogen. Carcinogenic effects observed in one long-term study with rats
probably were caused by the specific study conditions which resulted in persistent induction of liver
enzymes and/or deficiency of choline. However, it cannot be completely ruled out that thehepatotoxic effects caused by high and chronic doses of BHT may result in persistent cellproliferation, which is known as a possible mechanism of non-genotoxic carcinogens. In addition,
depending on the application regime, BHT may exert either anticarcinogenic or tumour-promoting
activity at relatively high doses. For the possible carcinogenic and tumour-promoting effect ofBHT, a threshold level of 100 mg/kg bw/day can be assumed based on the results from the study ofOlsen et al. with chronic BHT exposure starting in utero as a worst case scenario (no increase inliver carcinoma but slight increase in liver adenoma at 100 mg/kg bw/day; NOEL of this study at 25
mg/kg bw/day). From the carcinogenicity studies with rats and mice an overall NOAEL can not bedirectly derived.
UNEP PUBLICATIONS

21

OECD SIDS
3.1.8

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Toxicity for Reproduction

Effects on Fertility
In a three-generation study (Tanaka et al. 1993), Crj:CD-1 mice (F0 and F1 generations) received ca.
0, 23, 68, 203 or 608 mg/kg bw/day BHT in the diet during premating, mating, gestation andlactation periods (ca. 11 weeks). There were no effects on the number of litters, number of pups,
litter size, litter weight and sex ratio in any dose group of F1 and F2 animals or on neurobehaviouralparameters in F1 and F2 generation. The body weight of pups was increased at the lowest dose atbirth and during lactation period for each generation.
In the two-generation oral carcinogenicity study with Wistar rats (Olsen et al. 1986) discussed
above (see chapters 3.8 and 3.10), the NOAEL for maternal toxicity was 100 mg/kg bw/day; bodyweight gain of the F0 animals was significantly reduced in the high-dose group (500 mg/kg
bw/day). Gestation rate was not affected by the treatment. The Armitage-Cochran test for linear
trend in proportions demonstrated that the fraction of litters with ten or more pups decreased
significantly with BHT dose (P < 0.001). bw/dayAt weaning, these pups, particularly males,
showed a dose-related reduction of body weight being clearly statistically significant at 100 and 500
mg/kg bw/day.. The findings of the other two-generation feeding study with Wistar rats (Price
1994; McFarlane et al. 1997; see chapters 3.7 and 3.10) revealed slightly less maternal toxicity(NOAEL parental: females 500; males 100 mg/kg bw/day). Consequently, the NOEL for pup bodyweight and development (weight gain reduction during lactation) was higher, i.e. 100 mg/kg
bw/day, than in the above study. No adverse effects on reproductive performance were observed.
Conclusion
The only effects on reproduction were lower numbers of litters of ten or more pups at birth at dosesof 100 mg/kg bw/day and above. The NOAEL was 25 mg/kg bw/day in the rat.
Developmental Toxicity
Two teratogenicity tests were carried out with JCL-ICR mice (Tokyo Metropolitan ResearchLaboratory of Public Health 1978). After daily oral administration of 0, 70, 240 and 800 mg
BHT/kg bw/day between day 7 and 13 of gestation maternal toxicity was confined to increased
spleen weight and decreased kidney weight in the high-dose group. Reproductive and fetalparameters did not show any effects in all dose groups. The second test with a single administrationof 1200 or 1800 mg/kg bw on day 9 of gestation also revealed no teratogenic potential of BHT. The
only changed parameter was a delayed progression of ossification in the fetuses, which wasprobably due to the maternal toxicity of these high doses, as indicated by 10% and 25% mortality,
and increased spleen and lung weights of the dams. Thus, the NOEL for developmental toxicity was
800 mg/kg bw.
Two gavage studies with rats support the above findings. After daily application of up to 400 mg
BHT/kg bw/day to Sprague-Dawley rats between day 7 and 17 of gestation no evidence ofcompound-related fetal malformation was observed (Han et al. 1993). In a similar study (Tanaka etal. 1990), 2,2'-methylenebis (4-methyl-6-tert-butylphenol) was administered in doses up to 375
mg/kg bw to Wistar rats. At this dose, maternal toxicity and a slight increase in fetal mortality wasobserved. No teratogenic effects were seen. This compound is also used as an antioxidant andconsists essentially of two molecules of BHT.
Conclusion
During pregnancy BHT had maternal effects on mice above oral doses of 240 mg/kg bw/day. The
NOEL for developmental toxicity was 800 mg/kg bw/day.
22

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Other Toxicological Endpoints
There are a number of studies which have investigated the possible lung toxicity of repeated highdoses of BHT to mice. Obviously long-term dosages of 450 mg/kg bw/day and above lead to lungdamage (BUA, 1991).

UNEP PUBLICATIONS

23

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

4

HAZARDS TO THE ENVIRONMENT

4.1

Aquatic Effects

Acute Toxicity Test Results
Testing of the ecotoxicological behavior of BHT exhibits difficulties due to its extremely low watersolubility of about 0.6 to 1.1 mg/l at room temperature and its tendency to decompose to various
degradation products depending on the actual conditions (see chapt. 2.1.2). Based on the instability,
low recovery rates of applied BHT from the test solutions are not surprising. Due theaforementioned instability of BHT the data obtained in testing cover not only the toxicity of BHT
but also the toxicity of the oxidation/degradation products of BHT.
For the acute toxicity of BHT in aquatic species reliable experimental results from short-term tests
with fish, daphnia, algae and microorganisms are available. Only those effect values wereconsidered for the assessment that did not exceed the low water solubility of the compound (0.6 1.1 mg/l) and were based on measured concentrations.
A test on the acute toxicity of BHT to fish was conducted according to the European protocol EEC
C.1 (equivalent to OECD guideline 203). Semistatic exposure (renewal of test solution every 24
hours) of Brachydanio rerio in a limit test with water-saturated concentration of the test substance
had no adverse effect within the test period of 96 hours. The BHT concentration measured in thetest solution after 24 hours of exposure was 0.57 mg/l (Bayer AG 1994).
In a test according to the European protocol EEC C.2 (equivalent to OECD guideline 202, part 1)
the acute toxicity of BHT in invertebrates was determined. Daphnia magna were exposed in a limit
test with water-saturated concentration of the test substance. No toxic effects were observed during
incubation and the 48h EC0 based on the measured BHT concentration (geometric mean of TS
concentrations measured at the start and after 48 h of exposure) was reported to be ≥ 0.17 mg/l
(Bayer AG 1994).
A further acute test with Daphnia pulex is also available for BHT (Passino, Smith, 1987). This test
was performed according to ASTM method. Acetone was used as solvent with a concentration of
0.5 ml/l. A 48h EC50 of 1.44 mg/l related to nominal concentration was found. However theconcentration of acetone in this study was higher than proposed by OECD, US-EPA and EU (0.1ml/l). As there are other valid tests performed without solvent available that are regarded to be more
relevant, the test is not used for the effect assessment of BHT.
In a long-term test Daphnia magna were exposed to three BHT concentrations (0.1 mg/l; 0.316
mg/l and 1.0 mg/l; nominal) for a test duration of 21 days. The test was performed according toOECD guideline 202 (part 2) under semistatic conditions. The NOEC (endpoint: reproduction)
based on measured test substance concentrations (geometric mean of TS concentrations measured at
the start and after 48 h and 72 h of exposure at water exchange) was reported to be 0.07 mg/l (BayerAG 1994).
The acute toxicity of BHT to algae was determined in a test according to the European protocol
EEC C.3 (equivalent to OECD guideline 201). The saturated BHT-water concentration was tested
in a limit test with the algae Scenedesmus subspicatus. Only a slightly lower growth rate wasobserved after 72 h of incubation (8 % inhibition). Based on measured concentrations (geometricmean of TS concentrations measured at the start and after 72 h of exposure) an ErC8 of 0.4 mg/l isderived (Bayer AG 1994). This value can be used as a NOEC.

24

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

In a test with activated sludge according to Directive 88/302/EEC, Part C (respiration inhibitiontest) a 3h EC0 = 1000 mg/l was determined (Bayer AG 2000).
For the protozoan species Tetrahymena pyriformis a 24h EC50 of 1.7 mg/l was found in a cell
multiplication inhibition test (Yoshioka et al.,1985).
The lowest available long-term NOEC of 0.07 mg/l, found in a 21 days reproduction test with
Daphnia magna is used for the derivation of the PNECaqua. The application of an assessment factorof 50 is justified, as results from two long-term tests (daphnia and algae) are available, resulting in a
PNECaqua of 0.0014 mg/l.
4.2

Terrestrial Effects

No reliable data available.
4.3

Other Environmental Effects

In a feeding study with "White Leghorn" chicken (Gallus domesticus) the influence of BHT on
aflatoxin toxicity has been studied. 0.1 % BHT in the diet (8x the standard BHT concentration of
0.013 % in feed) had no significant inhibiting effect on weight gain, whereas 0.4% (30 x thestandard BHT concentration in feed) resulted in a temporary depression (36 %) of weight gain fromday 1 until 3 weeks; from week 3 to week 6 the weight gain was normal (98 % of control) and at theend of BHT treatment (6 weeks) the total weight gain amounted to 78 % of control. Both BHT
dosages improved body weight gain and feed efficiency of chicken treated with 3000 ppb aflatoxinin feed (Larsen et al., 1985).

UNEP PUBLICATIONS

25

OECD SIDS

52,6-DI-TERT-BUTYL-P-CRESOL (BHT)

CONCLUSIONS

Production and use:
The world production capacity of BHT amounts to about 62,000 t/a by more than 20 producers.
BHT is a registered antioxidant, licensed for food products, animal feed, cosmetics, and packaging
material. It is also used in petroleum products, synthetic rubbers, plastics, elastomers, oils, waxes,
soaps, paints, and inks.
Emission data from production of BHT in the sponsor country are available for Bayer AG.
Monitoring data at the outlet of the industrial biological waste water treatment plant lead to a worst
case PEC for the receiving water of < 0.003 µg/l. During normal operation no BHT is emitted into
the atmosphere. A significant exposure to the terrestrial compartment could not be identified.
Releases into the environment from application of BHT are not readily available.
A significant release of into the environment is expected from migration of BHT onto the surface of
products containing the substance.
Environmental behavior:
According to a Mackay Level I model calculation BHT is mainly distributed to air (79.3-87.5 %)followed by soil (6.1-10.2 %) and sediment (5.7-9.5 %). The calculated Henrys’ law constantindicates rapid volatilization from aqueous solution. Due to the instability of BHT in aqueous
solution both estimations reflect a tendency for BHT distribution among environmental
compartments.
BHT is relatively unstable under environmental conditions. Extent and products of decomposition
are dependent on several factors like irradiation, pH, temperature, moisture, presence of soil andsoil microorganisms, and oxygen content. In air BHT is rapidly photodegraded via reaction withhydroxyl radicals (t1/2 = 7.0 hours). In aqueous solution BHT is decomposed with and without
irradiation: Under conditions of natural sun light only 25 % of applied BHT was recovered after 8days of exposure, even in the corresponding test conducted in the dark about 40% of applied BHT
was altered to various degradation products. BHT is unstable in soil, too. Already within one day of
incubation in non-sterilized soils about 63-82 % BHT were decomposed. In sterilized soils 25-35 %of applied BHT were decomposed while 57-68 % remained unchanged. Under non-sterilized
conditions appreciable amounts of BHT (21-29 %) were mineralized to CO2. In a further
experiment in which the distribution of radioactivity between the compartments water, soil and fish
was measured in a model ecosystem, it was found that in the experiments where 14C-BHT was
directly applied to soil, between 62 and 74 % of the applied radioactivity remained in soil, 9 – 32 %was dissolved into water and only a very small part was found in fish. Volatilization was only ofminor importance. In the experiment where BHT was given to the water, about 30 % of applied
radioactivity remained in water, 5 to 14 % was adsorbed to soil and 3 to 10 % was translocated infish. More than 50 % was evaporated into atmosphere.
Depending on the exposure pathways, the compartments air, hydrosphere and soil can beenvironmental target compartments for this substance and its metabolites.
BHT is not readily biodegradable (4.5 % degradation after 28 days) in surface water and wastewater
treatment plants. The available data on bioaccumulation in aquatic species are inconsistent. A wide
range of BCF values between 2 and 2500 for fish, snails and algae have been reported.
Summarizing, it can be assumed that BHT has a moderate to high bioaccumulation potential.

26

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

For the toxicity of BHT on aquatic species reliable experimental results from tests with fish,
daphnia, algae and microorganisms are available. Only those reported effect values were considered
for the assessment that did not exceed the low water solubility of the compound (0.6 - 1.1 mg/l) andwere based on measured test substance concentrations.
In acute toxicity tests 96h LC0- and 48h EC0 values of ≥ 0.57 and ≥ 0.17 mg/l were obtained in fish
(Brachydanio rerio) and invertebrates (Daphnia magna), respectively. The 72h ErC8 in acutetoxicity testing in algae (Scenedesmus subspicatus) was determined to be = 0.4 mg/l. All values are
based on concentrations of BHT measured at the beginning of the test and at appointed times
thereafter. In a test with activated sludge a 3h EC0 1000 mg/l was obtained and in a test with theprotozoan species Tetrahymena pyriformis a 24h EC50 of 1.7 mg/l was found. In a 21 days
reproduction test with Daphnia magna a NOEC = 0.07 mg/l was determined based on measured
concentrations. Using an assessment factor of 50 a PNECaqua = 0.0014 mg/l is derived from thisvalue.
Human Health:
BHT caused acute toxic effects in mammals. In rats, the oral LD50 was > 2930 mg/kg bw, the LD50
after dermal exposure was > 2000 mg/kg bw. It was slightly irritating to the skin and eyes ofrabbits.
On chronic oral exposure of rats, liver and thyroid are the main targets. Doses above 25 mg BHT/kg
bw/day resulted in thyroid hyperactivity, enlargement of the liver, and induction of several liver
enzymes. 25 mg BHT/kg bw/day can be considered as NOAEL for chronic exposure. The
haemorrhagic effects of high repeated doses of BHT seen in certain strains of mice and rats, but notin other species, may be related to its ability to interact with prothrombin and vitamin K.
BHT is not a genotoxic carcinogen. Carcinogenic effects observed in one long-term study with rats
probably were caused by the specific study conditions. However, it cannot be completely ruled outthat the hepatotoxic effects caused by high and chronic doses of BHT may result in persistent cellproliferation, which is known as a possible mechanism of non-genotoxic carcinogens. In addition,
depending on the application regime, BHT may exert either anticarcinogenic or tumour-promoting
activity at relatively high doses. For the possible carcinogenic and tumour-promoting effect ofBHT, a threshold level of 100 mg/kg bw/day can be assumed based on the results from the study ofOlsen et al. with chronic BHT exposure starting in utero as a worst case scenario (no increase inliver carcinoma but slight increase in liver adenoma at 100 mg/kg bw/day; NOEL of this study at 25
mg/kg bw/day)
BHT showed no potential to cause point mutations in several Ames and other bacterial tests andmammalian test systems.
Overall, the available studies demonstrate that BHT has no clastogenic activity in vitro or in vivo.
Most in vitro chromosome aberration assays were negative as were sister chromatid exchange
assays and DNA damage and repair assays. In vivo, micronucleus assays with mice, cytogenetic
assays with rats and mice, dominant lethal assays with rats and mice, and the heritable translocation
assay with mice were also negative.
The only effects on reproduction were lower numbers of litters of ten or more pups at birth at dosesof 100 mg/kg bw/day and above. The NOEL was 25 mg/kg bw/day.
From studies with mice and rats there is no evidence of teratogenic effects of BHT. During
pregnancy BHT led to maternal effects on mice above oral doses of 240 mg/kg bw/day. The NOEL
for developmental toxicity was 800 mg/kg bw/day.

UNEP PUBLICATIONS

27

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Despite of being in wide dispersive use as ingredient of various products for many years only very
few cases of allergic reaction in humans after dermal exposure or oral intake have been described.
For the use of BHT as antioxidant in foodstuff an acceptable daily intake (ADI) of 0 - 0.3 mg/kg
bw/day has been established.

28

UNEP PUBLICATIONSOECD SIDS

62,6-DI-TERT-BUTYL-P-CRESOL (BHT)

RECOMMENDATIONS

Environment: The substance is a candidate for further work. Releases into the environment during
use of BHT and from products containing the substance have to be assumed but are notquantifiable. In the environment, BHT is rapidly decomposed forming several, partly unidentified,metabolites. BHT is not readily biodegradable, a moderate to high bioaccumulation potential has tobe assumed. The NOEC from the long-term toxicity to daphnids was 0.07 mg/l, resulting in a
PNEC of 0.0014 mg/l. Therefore, the performance of an environmental risk assessment isrecommended. Especially the questions concerning exposure, bioaccumulation as well as toxicity ofthe metabolites should be clarified.
Human Health: No recommendation for further work, because all SIDS endpoints are adequately
covered and because exposure is controlled in occupational settings.

UNEP PUBLICATIONS

29

OECD SIDS

7

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

REFERENCESACGIH = American Conference of Governmental Industrial Hygienists Inc., Cincinnati, Ohio:
Documentation of the threshold limit values and biological exposure indices: 5th ed. (1986), p. 227
Allen, J.R., Engblom, J.F. (1972): Food Cosmet. Toxicol. 20, 769-779
Bayer AG (2001): IUCLID Data Set 2,6-di-tert-butyl-p-cresol (CAS No. 128-37-0)
Bayer AG 1973, Internal Study, Test on density, AP-Nr. 514 497 (1973-03-09)
Bayer AG 1986a, Internal Study, Test on vapour pressure
Bayer AG 1986b, Internal Study, Test on water solubility
Bayer AG 1994, Internal Study, Tests on ecotoxicological behaviour of BHT - Acute tests on fish,
Daphnia, and alge, Chronic test on Daphnia; Report 466 A/94
Bayer AG 2000, Interne Untersuchungen zur Schadwirkung von BHT gegenüber Belebtschlamm;
report 1060 A/00
Bayer AG 2001, Personal communication, December 2001
Bayer AG 2001a, Calculation of the Henry's law constant of BHT according to HENRYWIN, v.
3.10 (22.11.2001)
Bayer AG 2001b+c, Calculation of the environmental distribution of BHT according to Mackay
fugacity model level I. Method published in: Mackay, D., Multimedia Environmental Models: The
Fugacity Approach. Lewis Publ. Inc., Michigan, U.S.A. (1991)
Bayer AG 2001d, Calculation of the Photodegradation of BHT according to AOPWIN, v. 1.90
(2000)
Bayer AG, 2002; personal communication, January 2002
Bomhard, E. (1996): J. Am. Coll. Toxicol. 15, S72
Bomhard, E.M. et al. (1992): Mutat. Res. 277, 187–200
Botterweck, A.A.M. et al. (2000): Food Chem. Toxicol. 38, 599–605
Bruce, W.R., Heddle, J.A. (1979): Can. J. Genet. Cytol. 21, 319–334
BUA (1991): BUA-Stoffbericht 58 – Butylhydroxytoluol (2,6-Bis(1,1-dimethylethyl)4-methylphenol), Beratergremium für umweltrelevante Altstoffe (BUA), VCH Weinheim
Chang, S.-S. & Maurey, J.R., J. Chem. Eng. Data 30, 384-387 (1985)
Cumming, R.B. et al. (1976): ORNL Biology Div. Annu. Rept., ORNL-5195, June 30, 20-22
de Boer, E.M. et al. (1989): Contact Dermatitis 20, 280–286
Dean, B.J. (ed.) (1983): Report of the United Kingdom Environmental Mutagen Society (UKEMS)
subcommittee on guidelines for mutagenicity testing. Part 1: Basic test battery; minimal criteria;
professional standards; interpretation; selection of supplementary assays. Chapter 7: Dominantlethal mutation assays, 143–164.
Deichmann, W.B. et al. (1955): Arch. Ind. Health 11, 93-101
30

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

DFG (1985): Toxikologisch-arbeitsmedizinische Begründung von MAK-Werten
Butylhydroxytoluol (BHT), Deutsche Forschungsgemeinschaft (DFG), VCH Weinheim–

EEC (1987): Commission Directive of 18 November 1987 adapting to technical progress for the
ninth time Council Directive 67/548/EEC on the approximation of laws, regulations andadministrative provisions relating to the classification, packaging and labelling of dangerous
substances. Official Journal of the European Communities 31, 76–78.
Flyvholm, M.–A., Menne, T. (1990): Contact Dermatitis 23, 341–345
Galloway, S.M. et al. (1987): Environ. Mol. Mutagen. 10, Suppl. 10, 1 - 175
Geyer, H. et al., Regul. Toxicol. Pharmacol. 6, 313-347 (1986)
Goh, C.L., Ho, S.F. (1993): Contact Dermatitis 28, 134–138
Goodman, D.L. et al. (1990): J. Allergy Clin. Immunol. 86, 570–575
Grillo, C.A., Dulout, F.N. (1995): Mutat. Res. 345, 73–78
Grogan, M.W.A. (1986): West. J. Med. 145, 245–246
Han, S.Y. et al. (1993): Teratology 48, 507, B–39
Hazleton France (1988a): Rapport No. 801300 to Rhone–Poulenc S.A.
Hazleton France (1988b): Rapport No. 801301 to Rhone–Poulenc S.A.
Heil, J. et al. (1996): Mutat. Res. 368, 181–194
Hirose, M. et al. (1981): Food Cosmet. Toxicol. 19, 147-151
IARC (1986): Monograph on the evaluation of the carcinogenic risk of chemicals to humans 40,
161–206
Inai, K. et al. (1988): Jpn. J. Cancer Res. (Gann) 79, 49–58
Inui, H. et al., Chemosphere 6, 383-391 (1979a)
Inui, H. et al., Chemosphere 6, 393-404 (1979b)
Kanerva, L. et al. (1997): Contact Dermatitis 37, 301–302
Kanerva, L. et al. (1999): Acta Derm. Venereol 79, 296–300
Larsen, C. et al. (1985): Poultry Science, 64, 2287-2291
LFU = Landesanstalt für Umweltschutz Baden Württemberg, Personal communi-cation to BUA(1994)
Mallette, F.S., von Haam, E. (1952): A.M.A. Arch. Ind. Hyg. Occup. Med. 5, 311–317
Maxwell, W.A. and Newell, G.W. (1974): Screening techniques for environmental mutagens. In:
Molecular and Environmental Aspects of Mutagenesis (Prakash, L., Sherman, F., Lawrence, C.W.
& Taber, H.W., eds.), Charles C. Thomas Publisher, Springfield, Illinois/USA; Chapter 14, pages
223-252
McFarlane, M. et al. (1997): Food Chem. Toxicol. 35, 753–767
McGregor, D.B. et al. (1988): Environ. Mol. Mutagen. 11, 91 - 118
UNEP PUBLICATIONS

31

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

Mikami, N. et al., Chemosphere 5, 305-310 (1979b)
Mikami, N. et al., Chemosphere 5, 311-315 (1979a)
MITI 1992, Biodegradation and Bioaccumulation Data of Existing Chemicals Based on the CSCL
Japan, Compiled under the Supervision of Chemical Products Safety Division, Basic Industries
Bureau MITI, Ed. by CITI, October 1992. Published by Japan Chemical Industry EcologyToxicology & Information Center
Miyakawa, Y. et al. (1986): Toxicol. Lett. 34, 99–105
NCI (1979) National Cancer Institute: NTIS/PB 298539
Newell G.W., Maxwell W.A. (1972): Stanford Research Institute, Project LSU-1348 for FDA U.S.
Dept. of Commerce, Services, NTIS PB-221827
OECD (1984): Guideline 478 "Genetic toxicology: rodent dominant lethal test". OECD Guidelines
for Testing of Chemicals
Olsen, P. et al. (1986): Food Chem. Toxicol. 24, 1–12
Pardee, W.A.; Weinrich, W.: Physical Properties of alkylated phenols. Ind. Eng. Chem. 36: 595–
603 (1944)
Paschin et al. (1986): Food Chem. Toxicol. 24, 881-883
Paschin, Y.V. & Bahitova, L.M. (1984): Mutation Res. 137, 57-59
Powell, C.J. et al. (1986): Food Chem. Toxicol. 24, 1131–1143
Price, S.C. (1994): Robens Institute; Report No. RI93/TOX/0020, 29 July 1994
Shell Research Ltd. 1983, Internal Study, Determination of the n-octanol/water partition coefficient
using a reverse-phase HPLC method
Sheu, C.W. et al. (1986): Environ. Mutagen. 8, 357–367
Shlian, D.M., Goldstone, J. (1986): N. Engl. J. Med. 314, 648–649
Spanjers, M.T., Til, H.P. (1978): Determination of the acute oral toxicity of Vulkanox KB in rats.
Unpublished report to Bayer AG, January 27, 1978
Takahashi, O. (1986): Arch. Toxicol. 58, 177-181
Takahashi, O. (1988): Arch. Toxicol. 62, 325-327
Takahashi, O. (1992): Food Chem. Toxicol. 30, 89-97
Takahashi, O. et al. (1980): Food Cosmet. Toxicol. 18, 229-235
Takahashi, O., Hiraga, K. (1978a) Toxicol. Appl. Pharmacol. 43, 399–406
Takahashi, O., Hiraga, K. (1978b): Food Cosmet. Toxicol. 16, 475–477
Takahashi, O., Hiraga, K. (1979): J. Nutr. 109, 453-457
Tanaka, S. et al. (1990): Eisei Shikenjo Hokoku 108, 52–57
Tanaka, T. et al. (1993): Toxicol. Lett. 66, 295–304
32

UNEP PUBLICATIONSOECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

The Merck Index 1983, Whitehouse Station, NJ, USA: Windholz M. Merck and Co., Inc.(ed), 215216, No. 1521: Butylated Hydroxytoluene
Thomas, R.G, Volatilization from water. In: Handbook of chemical property estimation methods;
Lyman, W.J., Reehl, W.F., Rosenblatt, D.H. (Eds.), McGraw-Hill Book Company, New York, p1516 (1990)
Tokyo Metropolitan Research Laboratory of Public Health (1978): In: Shell Oil Co. (1992):
NTIS/OTS 0535892
Watanabe, K. et al. (1998): Mutat. Res. 416, 169–181
WHO (1996): Toxicological evaluation of certain food additives and contaminants, 44th Meeting of
the Joint FAO/WHO Expert Committee on Food Additives (JECFA), WHO Food Additives Series
35, 3-86
Williams, G.M. et al. (1990a): Food Chem. Toxicol. 28, 799–806
Williams, G.M. et al. (1990b): Food Chem. Toxicol. 28, 793–798
Williams, G.M. et al. (1999): Food Chem. Toxicol. 37, 1027-1038
Yoshioka, Y. et al., Testing for the Toxicity of Chemicals with Tetrahymena pyriformis, The
Science of the Total Environment, 43, 149-157 (1985)

UNEP PUBLICATIONS

33

OECD SIDS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)

IUCLID
Data Set
Existing Chemical
CAS No.
EINECS NameEC No.
TSCA Name
Molecular Formula

:
:
:
:
:
:

ID: 128-37-0
128-37-0
2,6-di-tert-butyl-p-cresol
204-881-4
Phenol, 2,6-bis(1,1-dimethylethyl)-4-methylC15H24O

Producer related part
Company
Creation date

: Bayer AG: 03.03.1994

Substance related part
Company
Creation date

: Bayer AG: 03.03.1994

34

StatusMemo:: X AKTUELL EG / ICCA

Printing date
Revision date
Date of last update
Number of pages: 12.01.2004:: 12.01.2004: 1

Chapter (profile)
Reliability (profile)
Flags (profile): Chapter: 1, 2, 3, 4, 5, 6, 7, 8, 10: Reliability: without reliability, 1, 2, 3, 4
: Flags: without flag, non confidential, WGK (DE), TA-Luft (DE), Material
Safety Dataset, Risk Assessment, Directive 67/548/EEC, SIDSUNEP PUBLICATIONSOECD SIDS
1. GENERAL INFORMATION

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

1.0.1

APPLICANT AND COMPANY INFORMATION

1.0.2

LOCATION OF PRODUCTION SITE, IMPORTER OR FORMULATOR

1.0.3

IDENTITY OF RECIPIENTS

1.0.4

DETAILS ON CATEGORY/TEMPLATE

1.1.0

SUBSTANCE IDENTIFICATION

1.1.1

GENERAL SUBSTANCE INFORMATION

Purity type
Substance typePhysical status
PurityColour
Odour

:
:
:
:
:
:

Flag
03.12.2001:

organic
solid>= 99.8 % w/w

Critical study for SIDS endpoint

1.1.2

SPECTRA

1.2

SYNONYMS AND TRADENAMES

2,6-DI-TERT-BUTYL-4-METHYLPHENOL
Flag
20.05.1994:

Critical study for SIDS endpoint

2,6-DI-TERT-BUTYL-P-CRESOL
Flag
20.05.1994:

Critical study for SIDS endpoint

4-HYDROXY-3,5-DI-TERT-BUTYLTOLUENE
Flag
05.05.1994:

Critical study for SIDS endpoint:

Critical study for SIDS endpoint

BHT
Flag
19.05.1994

BUTYLATED HYDROXY TOLUENE

UNEP PUBLICATIONS

35

OECD SIDS
1. GENERAL INFORMATION

Flag
19.05.1994:

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Critical study for SIDS endpoint

BUTYLATED HYDROXYTOLUENE
Flag
05.05.1994:

Critical study for SIDS endpoint

P-CRESOL, 2,6-DI-TERT-BUTYLFlag
19.05.1994:

Critical study for SIDS endpoint

PHENOL, 2,6-BIS(1,1-DIMETHYLETHYL)-4-METHYLFlag
19.05.1994
1.3:

Critical study for SIDS endpoint

IMPURITIES

PurityCAS-NoEC-No
EINECS-Name
Molecular formula
Value

:
:
:
:
:
:

67-56-1
200-659-6
methanol<= .2 % w/w

03.12.2001
1.4

ADDITIVES

Remark19.05.1994

:

none

Remark

:Flag
30.11.2001:

World wide production amounts to about 62,000 t/a by morethan 20 producers in year 2000
Critical study for SIDS endpoint

Remark:no official labelling required

Flag
01.02.2002:

For food applications, substance requires E 321 notation.
Critical study for SIDS endpoint

1.5

1.6.1

36

TOTAL QUANTITYLABELLING

UNEP PUBLICATIONSOECD SIDS
1. GENERAL INFORMATION
1.6.2

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

CLASSIFICATION

Remark
Flag
01.02.2002::

no official classification required
Critical study for SIDS endpoint

Type of use
Category::

industrial
Fuel industry

Flag
16.08.2001:

Critical study for SIDS endpoint

Type of use
Category::

type
Wide dispersive use

Flag
16.08.2001:

Critical study for SIDS endpoint

Type of use
Category::

industrial
Polymers industry

Flag
19.05.1994:

Critical study for SIDS endpoint

Type of use
Category::

industrial
other: foodstuffs and feed industry

Flag
19.05.1994:

Critical study for SIDS endpoint

Type of use
Category::use
Food/foodstuff additives

Flag05.05.1994:

Critical study for SIDS endpoint

Type of use
Category::use
Stabilizers

Flag05.05.1994:

Critical study for SIDS endpoint

1.6.3

PACKAGING

1.7

USE PATTERN

16.08.2001
1.7.1

DETAILED USE PATTERNUNEP PUBLICATIONS

37

OECD SIDS
1. GENERAL INFORMATION

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

1.7.2

METHODS OF MANUFACTURE

1.8

REGULATORY MEASURES

1.8.1

OCCUPATIONAL EXPOSURE LIMIT VALUES

Type of limit
Limit value::

TLV (US)10 mg/m3

Remark

:Flag
30.11.2001:

OEL in Germany: acc. to TRGS 900, 10 mg/m3 inhalable
fraction
Critical study for SIDS endpoint

1.8.2

ACCEPTABLE RESIDUES LEVELS

1.8.3

WATER POLLUTIONClassified byLabelled byClass of danger:::

KBwS (DE)
1 (weakly water polluting)

01.10.2001
1.8.4

MAJOR ACCIDENT HAZARDS

Legislation
Substance listed
No. in Seveso directive

:
:
:

no

:

no classification

24.05.1994
1.8.5

AIR POLLUTION

Remark24.05.1994
1.8.6

LISTINGS E.G. CHEMICAL INVENTORIES

1.9.1

DEGRADATION/TRANSFORMATION PRODUCTS

1.9.2

COMPONENTS

38

UNEP PUBLICATIONSOECD SIDS
1. GENERAL INFORMATION
1.10

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

SOURCE OF EXPOSURE

30.11.2001
1.11

ADDITIONAL REMARKS

1.12

LAST LITERATURE SEARCH

Type of search
Chapters covered
Date of search:::Internal and External

Remark

:Flag
31.01.2001:

Toxicology: April 1999
Environmental and ecotoxicology: April 2000
Critical study for SIDS endpoint

1.13

REVIEWSMemo:

BUA Report No. 58 (Butylated hydroxytoluene) and Suppl. No. 219Advisory Committee on Existing Chemicals, VCH Weinheim

Flag
31.01.2001:

Critical study for SIDS endpoint

UNEP PUBLICATIONS

39

OECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES

2.1

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

MELTING POINT

Value
Sublimation
MethodYear
GLP
Test substance

:
:
:
:
:
:

69.8 °Cnoother: differential scanning calorimetry
1985

Flag
14.01.2002:

Critical study for SIDS endpoint

Value

:

other TS: food grade, 99 % purity minimum

(1)
70 °C

13.11.2001
Value(2) (3) (4) (5):69 - 69.5 °C

13.11.2001
Value

(6)
:

70 - 71 °C

13.11.2001
2.2

(7)

BOILING POINT

Value

:

265 °C at 1013 hPa

Flag
10.08.2001:

Critical study for SIDS endpoint

Value

:

(2) (8) (3) (4) (5)266 °C at 1013 hPa

06.08.2001
2.3

(7)

DENSITY

Type
Value
MethodYear
GLP
Test substance

:
:
:
:
:
:

density
1.03 - 1.031 g/cm³ at 20 °C
other: helium compression pycnometer method
1973

Flag
30.11.2001:

Critical study for SIDS endpoint

Type
Value

:
:

(9)
density
1.048 at 20 °C

10.08.2001

40(8) (3) (5)UNEP PUBLICATIONSOECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES
2.3.1

GRANULOMETRY

2.4

VAPOUR PRESSURE

Value
Decomposition
MethodYear
GLP
Test substance

:
:
:
:
:
:

Method:Remark

:Flag
14.01.2002

:

Value

:

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004.011 hPa at 20 °C
Directive 84/449/EEC, A.4 "Vapour pressure"
1986

The vapour pressure was determined with a vapour pressure balance andby a dynamic procedure.
No further details are available.
The vapour pressure determined in this study is considered to be more
valid than the value of 0.02 hPa published in the "Auer Technikum
Ausgabe 12" of 1988 without any further explanation in terms of method
employed.
Critical study for SIDS endpoint(10).02 hPa at 20 °C

10.08.2001
Value

(8)
:

.16 hPa at 50 °C

10.08.2001

(8)

Value
Decomposition
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark

:17.10.2003
2.5.003 hPa at 25 °C
other (measured): vapour pressure balance
1987

Original reference not available.
No experimental details apart from the principle (vapour pressure balance)
available.
(11)

PARTITION COEFFICIENT

Partition coefficient
Log pow
pH value
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

Method:5.1 at °C
other (measured): HPLC method1983
yesother TS: 99 % purity minimum
Reverse-phase HPLC with a C18-coated silica gel column was used todetermine the retention time of the compounds investigated.
By using compounds with known log Kow values determined by theconventional "shake flask" method, it is possible to calibrate the HPLC
method and determine unknown log Kow values from retention times

UNEP PUBLICATIONS

41

OECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES

Flag
14.01.2002:

Partition coefficient
Log pow
pH value
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

Remark
Flag
14.01.2002::

Partition coefficient
Log pow
pH value
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

Remark:through the relationship between retention times on the HPLC column andknown log Kow values.
Critical study for SIDS endpoint(12)
5.03 at °C
other (calculated)
2001

Calculated with SRC-KowWIN v1.66 (2000)
Critical study for SIDS endpoint(13)

14.01.2002
Partition coefficient
Log pow
pH value
MethodYear
GLP
Test substance

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

:
:
:
:
:
:
:

4.17 at 37 °C
other (measured): spectrochemical detection
1979

The spectrophotometric determination used may encounter somedifficulties as the concentrations to be measured in the two liquid phases (ifabout equal volumes were used) differ by about 4 orders of magnitude andso some experimental error is possible.
(14)
6.2 at °C
other (calculated)

06.08.2001
2.6.1

SOLUBILITY IN DIFFERENT MEDIA

Solubility inValue
pH value
concentration
Temperature effects
Examine different pol.
pKa
Description
StableDeg. product
MethodYear
GLP
Test substance

42

(15)

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Water
1.1 mg/l at 20 °C
at °C
at 25 °C

Directive 84/449/EEC, A.6
1986
other TS: Merck-Schuchardt, 98 %UNEP PUBLICATIONSOECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Method:Remark
Flag
14.01.2002::

Solubility inValue
pH value
concentration
Temperature effects
Examine different pol.
pKa
Description
StableDeg. product
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Water
1.01 - 1.04 mg/l at 20 °C

Remark
Flag
14.01.2002::

water from a gravel pit
Critical study for SIDS endpoint

Solubility inValue
pH value
concentration
Temperature effects
Examine different pol.
pKa
Description
StableDeg. product
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Water
ca. 1.5 mg/l at 30 °C

Method:Flag
14.01.2002:

14-C-labeled BHT is mixed with distilled water for 4 days. The 14-C activity
quickly rises for the first 4 hours and slowly afterwards. The quick rise iscaused by the solution kinetics of BHT, the slow rise by BHT
decomposition. The extrapolation of 14-C activity levels from observations
after more than 4 hours to t = 0 results in the water solubility corrected forany decomposition in water.
Critical study for SIDS endpoint(1)

Solubility inValue
pH value
concentration
Temperature effects
Examine different pol.
pKa
Description
StableDeg. product

:
:
:
:
:
:
:
:
:
:

The solubility in distilled water was determined by HPLC. The solubility ingravel pit water was determined by mixing with BHT for several days andafterwards analysis without any further details.
solubility in distl. water
Critical study for SIDS endpoint
(16)

at °C
at 25 °C

Directive 84/449/EEC, A.6
1986
other TS: Merck-Schuchardt, 98 %

(16)

at °C
at 25 °C

other: radioactive labeled BHT
1985
other TS: food grade, 99 % purity minimum

Water.6 mg/l at 25 °C
at °C
at 25 °C

UNEP PUBLICATIONS

43OECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

MethodYear
GLP
Test substance

:
:
:
:

other: no data
1979

Method:Flag
17.10.2003:

Water solubility of 0.6 ppm is reported without further details, e.g. on themethod. When following the quotations, one gets to the papers "Mikami etal., Chemosphere 5: 311-315 (1979)" and "Mikami et al., Chemosphere 5:
305-310 (1979)" in which the determination of 14-C-labeled BHT is
described in brief as an extraction of water with ethyl acetate followed by
14-C- measurement in a liquid scintillation spectrometer. But even there no
more details are available.
Critical study for SIDS endpoint(17)

Solubility inValue
pH value
concentration
Temperature effects
Examine different pol.
pKa
Description
Stable

:
:
:
:
:
:
:
:
:

other TS: purity of radiolabelled BHT > 99 %Water
.4 mg/l at 20 °C
at °C
at 25 °C

01.10.2001
2.6.2

SURFACE TENSION

2.7

FLASH POINT

(18)

Value
Type

:
:

127 °C
open cup

Flag
03.08.2001:

Critical study for SIDS endpoint

2.8(8) (5)

AUTO FLAMMABILITY

MethodYear
GLP
Test substance

:
:
:
:

Directive 84/449/EEC, A.16 "Auto-flammability of solids"
2001
yesother TS: purity 99.9 %Result:Flag
14.01.2002:

substance does not undergo spontaneous combustion in thesense of EC Guideline A 16 (tested up to a temperature of
400 °C)
Critical study for SIDS endpoint

Remark:(19)

Ignition temperature: 345 °C
The ignition temperature is mentioned without any further explanation interms of method employed.

06.08.2001

44

(8)

UNEP PUBLICATIONSOECD SIDS
2. PHYSICO-CHEMICAL PROPERTIES

2.9

FLAMMABILITY

2.10

EXPLOSIVE PROPERTIES

Result:2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

other: class of dust explosion (DE): St2 (= danger of dust explosion)

01.10.2001

(20)

2.11

OXIDIZING PROPERTIES

2.12

DISSOCIATION CONSTANT

Acid-base constant

:

12.07

Flag
14.01.2002:

Critical study for SIDS endpoint

2.13

VISCOSITY

2.14

ADDITIONAL REMARKS(14)

UNEP PUBLICATIONS

45

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

3.1.1

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

PHOTODEGRADATION

Type:Light source:
Light spectrum:
Relative intensity:
DIRECT PHOTOLYSIS
Halflife t1/2:
Degradation:
Quantum yield:
INDIRECT PHOTOLYSIS
Sensitizer:
Conc. of sensitizer:
Rate constant:Degradation:
Deg. product:
Method
:
Year
:
GLP
:
Test substance:
Remark:Reliability:

Flag
15.01.2002:

Type
Light source
Light spectrum
Relative intensity
Conc. of substanceDeg. product
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Result:

Test condition

air
nm
based on intensity of sunlightca. 7 hour(s)% after
OH
1500000 molecule/cm³
cm³/(molecule*sec)% afterother (calculated): acc. to AOPWIN, v.1.90 (2000)
2001

Calculated half-life: t1/2 = 7.0 hours (1.5 x 10E6 OH
radicals/cm3: concentration during 12 daylight-hours acc.to
U.S.EPA;Rate constant kOH = 18.3 x 10E-12 cm3/molecule x s)(2) valid with restrictions
accepted calculation method
Critical study for SIDS endpoint
(21)

:

water
other: natural sun lightnm
based on intensity of sunlight.6 mg/l at °C
other (measured)
1977
no dataother TSa.) In distilled water 25.2% of applied radiolabelled BHT
was found after 8 days of exposure; as degradation products
were identified BHT-OOH (5.7%), BHT-OH (4.2%), BHT-CH2OH(7.5%), BHT-CHO (2.7%), and BHT-COOH (4.7%); polar
unidentified products amounted to ca. 48%.
b.) In the presence of soil and irradiation of test
solution, <0.1% of applied BHT could be recovered from thewater phase and <2.6% from soil. The sum of identified degradation
products of BHT amounted to <6.9% in water and <6.9% in soil after 30
days.
Bound 14-C in soil was <36.5% after 30 days.
a.) Test medium distilled water; test duration: 8 days, 8hours sunlight per dayb.) Photolysis in the presence of soil (light clay, sandy
clay loam, sandy loam); test duration: 30 days, 8 hours sunlight per day

46

UNEP PUBLICATIONSOECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS
Test substance
Reliability

:
:

Flag
15.01.2002

:

3.1.2

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

purity of 4-14 CH3-BHT > 99 %(2) valid with restrictions
study well documented, meets generally accepted scientificprinciples
Critical study for SIDS endpoint(22)

STABILITY IN WATERType
t1/2 pH4
t1/2 pH7
t1/2 pH9
Deg. product
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

Result:

Test condition:

Reliability:

Flag
15.01.2002:

Deg. product
MethodYear
GLP
Test substance

:
:
:
:
:

Method:abiotic
at °C
at °C
at °C
other: (measured)
no dataother TS: purity of radiolabelled BHT > 99%a.) In distilled water 59.6% of radiolabelled BHT wasrecovered after 8 days of exposure in the dark (volatiles
amounted to 0.2%); as degradation products were identified
BHT-OOH (2.6%), BHT-OH (8.8%), BHT-CH2OH (1.1%), BHT-CHO(3.0%), and BHT-COOH (1.4%); polar unidentified products
amounted to ca. 23%.
b.) In the presence of soil in the dark, <3% of BHT were detected in thewater phase and <7% in soil after 30 days of
exposure. Under these conditions 10-15% evaporated from the test
system.
The sum of identified degradation products amounted to <13% in the waterphase and <12.5% in soil.
Bound 14-C in the soil was <41% after 30 days.
a.) Test medium: distilled water without irradiation;
test duration: 8 days;b.) Test medium: aqueous solution of BHT in the presence ofdifferent soils (light clay, sandy clay loam, sandy loam);test duration: 30 days.
(2) valid with restrictions
study well documented, meets generally accepted scientificprinciples
Critical study for SIDS endpoint(22)
other: see remarks
other TS: 14C-phenyl-BHT > 99%
Stability of BHT was determined in a model ecosystem under
three different test conditions. The model ecosystem(aquarium) was separated into two compartments via a glass
plate with holes. In the larger compartment soil was placedon the bottom of the aquarium. The test solution was
circulated between the two compartments by a water pump.
A.) 14C-phenyl-BHT (5 ppm soil dw) was mixed with 500 g soil

UNEP PUBLICATIONS

47

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

and placed on the bottom of the aquarium; standard mineralsolution was added and circulated; the system was operated
for 4 weeks. BHT and its degradation products were measured(TLC) at appointed intervals.
B.) 500 g soil was spread on the bottom of the aquarium.
Water was added. After 1 day, 14C-phenyl-BHT (2 ppm) as anemulsion with Tween-80 (20 ppm) as solubilizing agent wasadded (water was sampled as in test A).

Result:C.) Two kg of soil treated with 14C-phenyl-BHT (2 ppm soildw) were placed in the aquarium. The ecosystem was operated
in a greenhouse for 4 weeks.
The distribution of radioactivity between the compartments water, soil andfish was measured at day 7, 14 and 28.
In the experiments A and C between 62 and 74 % of the applied
radioactivity remained in soil, 9 - 32 % was dissolved into water and only avery small part was found in fish. Recovery of 14C in the system was
between 85 to 97 % showing that volatilization was only of minor
importance.
In experiment B about 30 % of applied radioactivity remained in water, 5 to
14 % was adsorbed to soil and 3 to 10 % was translocated in fish.
Recovery of total 14C was 40 to 50 %, suggesting that more than 50 %was evaporated into atmosphere.
A.) BHT concentration in the water phase increased graduallytill day 18 (BHT release from soil). Afterwards BHT
concentration decreased. BHT-OH (rapid increase; decrease
after 4 days of exposure) and BHT-COOH (rapid increase
within the first days of exposure; then gradually increase
reaching a plateau) were detected as degradation products.
B.) In water, BHT concentration decreased to a minimum
concentration within 3 days mainly due to adsorption to soil
and evaporation (>50%). Thereafter concentration of BHT
gradually increased due to desorption of BHT from soil.
BHT-OH and BHT-COOH appeared gradually showing maximum
values and then decreased after about 14 days.

Test condition:

Reliability:

Flag
31.01.2002

:

3.1.3

(23)

STABILITY IN SOIL

TypeRadiolabel
Concentration
Soil temperature
Soil humidity

48C.) In the water phase the concentration of BHT increased
for the first 7 days of exposure, then decreased. The
concentration of BHT-OH was fairly constant. BHT-COOH
increased with time.
soil conditions: sand 80%, silt 12%, clay 8%, organic matter
1.8 %temperature: 25 +/- 2°C(2) valid with restrictions
Test design not aquivalent to current standard methods;
isolated results, however, are considered valid.
Critical study for SIDS endpoint

:
:
:
:
:

laboratory
yes
1 mg/kg
25 °C
40 other: % of maximum water-holding capacity

UNEP PUBLICATIONSOECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS
Soil classification
Year
Deg. product
Method

:
:
:
:

Year
GLP
Test substance:::

Result:2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

other: comparable to later OECD Guideline 304 A "Inherent
biodegradability in soil", 1981
1979
noother TS: >= 99%
Nonsterilized conditions:
After one day 63-82% of BHT were decomposed (about 1-2% mineralized
to CO2).
After 24 days of incubation 77-92% were decomposed (21-29%
mineralized to CO2).
Sterilized conditions:
After one day 25-35% of BHT were decomposed.
After 24 days of incubation 27-41% were decomposed. In both cases
mineralization was negligible (<2%).
After one day 57-68% of BHT and after 24 days 50-61% remained
unchanged.

Test condition:

Reliability:

Flag
16.01.2002

:

3.2.1

Under sterilized and nonsterilized conditions BHT-OOH, BHT-OH, BHTCH2OH, BHT-CHO, BHT-COOH were identified as degradation products of
BHT.
Radiolabeling: 14CH3-BHT
Natural soils tested:pHsand silt clay
light clay:
5.5
31% 40%29%
sandy clay loam: 6.3
65% 18%17%
sandy loam:
7.0
95%
3%
2%
soils adjusted to 40% of maximum water-holding capacity;
BHT concentration under sterilized and nonsterilized
conditions: 1 mg/kg; temperature: 25°C
(2) valid with restrictions
study well documented, meets generally accepted scientificprinciples
Critical study for SIDS endpoint(24)

MONITORING DATA

Type of measurement
Media
Concentration
Method::::background concentration
surface water

Result:

BHT concentrations determined in River Rhine at Karlsruheand Öhningen (Germany, Baden-Württemberg) during July December 1990 were consistently below the determination limit of 1 ug/l.
More refined measurements were conducted by the
Baden-Württemberg water authorities in River Rhine at
Mannheim, in River Neckar at Mannheim, and in River Donau at
Ulm during January - December 1991; the determination limit
was in all cases 0.02 ug/l:Rhine: <0.02-0.09 ug/l
50 percentile: < 0.02 ug/l

UNEP PUBLICATIONS

49

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

90 percentile: 0.08 ug/lDonau: <0.02-0.16 ug/l
50 percentile: 0.03 ug/l
90 percentile: 0.09 ug/l
Neckar: <0.02-0.09 ug/l
50 percentile: 0.02 ug/l
90 percentile: 0.08 ug/l(2) valid with restrictions
Results of monitoring programmes without detailed
documentation
Critical study for SIDS endpoint

Reliability:

Flag
15.01.2002:

Type of measurement
Media
Concentration
Method::::background concentration
surface water

Result:Reliability:

In 1992 and 1993, BHT concentrations in the river Rhine andhis tributaries were < 1 ug/l (determination limit: 1 ug/l)in Nordrhein-Westfalen (Germany). As a consequence thefurther monitoring was stopped.
(2) valid with restrictions
Results of monitoring programmes without detailed
documentation(25)

28.11.2001

(26)

3.2.2

FIELD STUDIES

3.3.1

TRANSPORT BETWEEN ENVIRONMENTAL COMPARTMENTS

50

Type
Media
Air
Water
Soil
Biota
Soil
Method
Year

:
:
:
:
:
:
:
:
:

volatility
water - air% (Fugacity Model Level I)% (Fugacity Model Level I)% (Fugacity Model Level I)% (Fugacity Model Level II/III)% (Fugacity Model Level II/III)other: calculation of Henry's law constant acc. to HENRYWIN v3.10 (2000)
2001

Remark:Result:

The two water solubilities (0.6 and 1.1 mg/l) used are those determined atabout environmentally relevant temperatures in distilled water marking theendpoints of the measured interval.
H = 220 Pa x m3/mol (at 20 °C)
based on a water solubility of 1.1 mg/l and a vapour
pressure of 1.1 Pa (see chapter 2.4 and 2.6.1)

Reliability

:

Flag

:

H = 404 Pa x m3/mol (at 20-25 °C)
based on a water solubility of 0.6 mg/l and a vapour
pressure of 1.1 Pa (see chapter 2.4 and 2.6.1)(2) valid with restrictions
accepted calculation method
Critical study for SIDS endpoint

UNEP PUBLICATIONSOECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

20.10.2003

(27)

Type
Media
Air
Water
Soil
Biota
Soil
Method
Year

:
:
:
:
:
:
:
:
:

adsorption
other: water-sediment% (Fugacity Model Level I)% (Fugacity Model Level I)% (Fugacity Model Level I)% (Fugacity Model Level II/III)% (Fugacity Model Level II/III)other: Field observation
1976

Method:ResultReliability::

Adsorption to river sediment calculated from measured mean
concentrations in river water and sediment; GC(FID), GC(ECD) and
GC/MS analysis
Derived adsorption factor: 4000
(3) invalid
Inadmissable derivation of adsorption factor: No data atequilibrium concerning the system sediment and water are
possible; no information about the composition of thesediment besides the statement "... varied from large gravel
to an organic-rich black ooze."

15.01.2002
3.3.2

(28)

DISTRIBUTION

Media
MethodYear:::

air - biota - sediment(s) - soil - waterCalculation according Mackay, Level I
2001

Remark:Result:

Test condition:

In terms of BHT adsorbed on aerosols the Junge equation (Technical
Guidance Documents in Support of the Commission Directive 93/67/EEC
on Risk Assessment for New Notified Substances and the Commission
Regulation (EC) 1488/94 on Risk Assessment for Existing Substances,
TGD) has to be considered: Only at vapour pressures near 0.0001 Pa an
appreciable part is adsorbed on aerosols, but at a vapour pressure of about
1 Pa (as reported for BHT at 20°C) the part adsorbed to aerosol isestimated at about 0.01 % of the total BHT in the atmosphere. So theaerosol as a whole can be ignored without producing any appreciable error.
Air:
87.5 %
Water:
0.6 %
Soil:
6.1 %
Sediment:
5.7 %suspended Sediment: <0.1 %Biota:<0.1 %
base data used (see chapter 2):
temperature [°C]:20
molar mass [g/mol]:
220.36
vapour pressure [Pa]:1.1
water solubility [mg/l]:
0.6
log Pow:
5.1
volumes used [m3]:air:
6 000 000 000
water:
7 000 000
soil:
45 000sediment:
21 000susp. sediment:
35.0
biota (fish):
7.0

UNEP PUBLICATIONS

51

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS
Reliability:

Flag
20.10.2003

:

Media
MethodYear:::

air - biota - sediment(s) - soil - waterCalculation according Mackay, Level I
2001

Remark:Result:

Test condition:

In terms of BHT adsorbed on aerosols the Junge equation (Technical
Guidance Documents in Support of the Commission Directive 93/67/EEC
on Risk Assessment for New Notified Substances and the Commission
Regulation (EC) 1488/94 on Risk Assessment for Existing Substances,
TGD) has to be considered: Only at vapour pressures near 0.0001 Pa an
appreciable part is adsorbed on aerosols, but at a vapour pressure of about
1 Pa (as reported for BHT at 20°C) the part adsorbed to aerosol isestimated at about 0.01 % of the total BHT in the atmosphere. So theaerosol as a whole can be ignored without producing any appreciable error.
Air:
79.3 %
Water:
1.0 %Soil:
10.2 %
Sediment:
9.5 %suspended Sediment: <0.1 %Biota:<0.1 %
base data used (see chapter 2):
temperature [°C]:20
molar mass [g/mol]:
220.36
vapour pressure [Pa]:1.1
water solubility [mg/l]:
1.1
log Pow:
5.1(2) valid with restrictions
accepted calculation method
Critical study for SIDS endpoint(29)

Reliability:

Flag
20.10.2003:

volumes used [m3]:air:
6 000 000 000
water:
7 000 000
soil:
45 000sediment:
21 000susp. sediment:
35.0
biota (fish):
7.0(2) valid with restrictions
accepted calculation method
Critical study for SIDS endpoint
(30)

3.4

MODE OF DEGRADATION IN ACTUAL USE

3.5

BIODEGRADATION

52

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type
Inoculum
Concentration:::

Contact time
Degradation
ResultDeg. product
Method
Year

:
:
:
:
:
:

aerobic
activated sludge
50 mg/l related to Test substance
related to
4.5 (±) % after 28 day(s)other: see remarks
1992

UNEP PUBLICATIONSOECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

GLP
Test substance::no datano data

Remark:Test condition:

Reliability:

Flag
21.10.2003:

The test was conducted in accordance with "Biodegradation
test of chemical substance by microorganisms etc."
stipulated in the Japanese Order Prescribing the Items of
the Test Relating to the New Chemical Substance (1974). This guideline
corresponds to OECD Guideline 301C, "Ready
Biodegradability: Modified MITI Test I" (1981). Degradation is given interms of BOD.
deviations from guideline:
sludge concentration: 50 mg/l
substance concentration: 50 mg/l(2) valid with restrictions
Test procedure comparable to guideline
Critical study for SIDS endpoint
(31)

Type
InoculumDeg. product
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

aerobic
activated sludge

Remark:Result:

Test condition:

Reliability:

Flag
15.01.2002:

Considering the very low solubility a series of tests werecarried out with radiolabeled BHT (14CH3- as well as
14C-phenyl-BHT) in different concentrations together withdifferent concentrations of activated sludge and with andwithout 0.2 ml/l ethanol in an aerated system. Only one test
set with BHT at saturation concentration (1 mg/l) and noethanol as suspension/solvent aid, but with 14CH3-BHT was
carried out and is regarded further for results.
According to the OECD Guidance Document on Aquatic Toxicity
Testing of Difficult Substances and Mixtures (June 2000),
the concentration of the solvent in the final test medium
should not exceed 0,1 ml/l.
Slow primary degradation (5.2 % 14CO2 of 14C-CH3-BHT), residual BHT
in solution (10.8 %) and volatilisation of BHT (26.2 %) after 35 days of
incubation is reported and regarded as a valid result. The percent values
respond to a total recovery of 14C of 82.9 %. Results of the other test sets
which are regarded as invalid due to a too high ethanol concentration, are
not reported here.
Incubation at 25°C in the dark, sludge concentration 3 ppm,
closed Erlenmeyer flask system with magnetic stirrer andCO2-free air supply (5 ml/min).
(2) valid with restrictions
Test design not equivalent to current standard methods;
isolated results, however, considered valid.
Critical study for SIDS endpoint
(17)

Type
Inoculum
Concentration:::

Contact time
Degradation
Result

:
:
:

other
1979
no dataother TS: purity of radiolabelled BHT > 99%

aerobic
predominantly domestic sewage
20 mg/l related to Test substance
related to< 10 (±) % after 20 day(s)UNEP PUBLICATIONS

53

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS
Deg. product
MethodYear
GLP
Test substance

:
:
:
:
:

RemarkReliability::

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

other: "geschlossener Flaschentest"
1978
noother TS: commercial sample
Emulgator W used as emulsifier(4) not assignable
documentation insufficient for assessment

30.11.2001
3.6

(32)

BOD5, COD OR BOD5/COD RATIO

Remark:ThOD: 2976 mg/g
CSB: 1704 mg/g
Above three parameters are extremly difficult to determine
because pure test substance BHT is sparingly soluble inwater, and other water solubles from excess BHT in water may
give rise to artefacts not related to BHT.

25.01.2001
3.7

54

(33)

BIOACCUMULATION

Species
Exposure period
Concentration
BCF
Elimination
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

Cyprinus carpio (Fish, fresh water)
56 day(s) at 25 °C.005 mg/l
330 - 1800

Remark:Reliability:

Flag
20.10.2003:

The test was conducted in accordance with "Bioaccumulation
test of chemical substance in fish and shellfish" stipulated in the JapaneseOrder Prescribing the Items of the Test Relating to the New Chemical
Substance (1974).
This guideline corresponds to OECD Guideline 305C,"Bioaccumulation: Degree of Bioconcentration in Fish" (1981)
The test is performed under flow through conditions, and test water isanalysed twice a week(2) valid with restrictions
guideline study; no explanation of the high variation oftest results is given, no details on analytical monitoring
Critical study for SIDS endpoint(31)

Species
Exposure period
Concentration
BCF
Elimination
Method
Year
GLP

:
:
:
:
:
:
:
:

other: see remarks
1992
no datano data

Cyprinus carpio (Fish, fresh water)
56 day(s) at 25 °C.05 mg/l
230 - 2500
other: see remarks
1992
no data

UNEP PUBLICATIONSOECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test substance:no data

Remark:Reliability:

Flag
20.10.2003:

The test was conducted in accordance with "Bioaccumulation
test of chemical substance in fish and shellfish" stipulated in the JapaneseOrder Prescribing the Items of the Test Relating to the New Chemical
Substance (1974).
This guideline corresponds to OECD Guideline 305C,"Bioaccumulation: Degree of Bioconcentration in Fish" (1981)
The test is performed under flow through conditions, and test water isanalysed twice a week.
(2) valid with restrictions
guideline study; no explanation of the high variation of
results is given; no details on analytical monitoring
Critical study for SIDS endpoint(31)

Species:

Exposure period
Concentration
BCF
Elimination
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

Remark:other: Cyprinus carpio (Fish, fresh water); Cypangopaludina japonica(snail); Daphnia pulex; algaeday(s) at 25 °C
1 - 125
yesother: see remarks
1979noother TS: 14C-phenyl-BHT > 99%
BCF was determined in a model ecosystem under threedifferent test conditions. The model ecosystem (aquarium)
was separated into two compartments via a glass plate withholes. In the larger compartment soil was placed on thebottom of the aquarium. The test solution was circulated
between the two compartments by a water pump.
A.) 14C-phenyl-BHT (5 ppm soil dw) was mixed with 500 g soil and placedon the bottom of the aquarium. Standard mineral solution was added andcirculated. After 4 days 2 carps were added to the smaller compartment.
The whole system was operated for 4 weeks. BHT and its degradation
products were measured (TLC) at appointed intervals. Carps and soil wereanalysed after 1, 2, and 4 weeks of exposure.
B.) 500 g soil was spread on the bottom of the aquarium.
Water and 2 carps were added. After 1 day, 14C-phenyl-BHT (2 ppm) as
an emulsion with Tween-80 (20 ppm) as solubilizing agent was added(water, fish, and soil were sampled as in test A).
C.) Two kg of soil treated with 14C-phenyl-BHT (2 ppm soildw) were placed in the aquarium. After 4 days, 2 carps wereadded to the smaller compartment, 2 snails (Cypangopaludina
japonica, 15-25 g), 1 g daphnids (Daphnia pulex), and 100 ml algal
suspension (filamentous green algae and Chlorella) were placed in thelarger compartment. The ecosystem was operated in a greenhouse for 4
weeks. Carps, snails, daphnids, and algae were analysed in a similar way
as in test A.
Although temperature during incubation is not reported, it is assumed thatthe tests were performed at 25 °C as this is the temperature used during
acclimatization of fish and in foregoing study.
The BCF values were derived from model ecosystems where adsorbant

UNEP PUBLICATIONS

55

OECD SIDS
3. ENVIRONMENTAL FATE AND PATHWAYS

Result:2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

materials (soil) was present.
There were only 2 fish in each experimental condition. It is not clear
whether this test was performed in a flow through system, and finally,
degradation of the substance is not taken into account.
A.) Only 1-2% of the applied radioactivity was translocatedin fish.
Exposure (days)
7
14
28
BCF (fish)
25
22
17
The BCF decreased with time. The same holds true for the
degradation products of BHT: BHT-OOH, BHT-OH, BHT-CH2OH,
BHT-CHO, and BHT-COOH. For the degradation products a
maximum BCF of 26 was determined after 7 days of exposure(BHT-OH). From the results the authors concluded that thecompounds in fish were excreted or metabolized fairly
rapidly.
B.) During this experiment ca. 50% of applied radioactivity
evaporated into the atmosphere.
Exposure (days)
7
14
28
BCF (fish)
16
21
13
The BCF values of the degradation products BHT-OH, BHT-CHO,
and BHT-OOH/BHT-COOH (for the last two compounds there are
discrepancies between the BCF value cited in the table andthe text) at the 7th day were very high: 347, 151, and 101,
respectively. The authors concluded that at the beginningfish incorporated BHT, then metabolized it before excretion(in the water phase BHT concentration decreased to a minimum value
within 3 days due to sorption of BHT to the soil).

Test condition:

Reliability:

Flag
12.01.2004

:

3.8

56

C.) During this experiment 15-18% of applied radioactivity
evaporated into the atmosphere.
Exposure (days)
7
14
28
BCF (fish)
15
13
2
BCF (snails)
91
125
30
BCF (daphnids)73
65 not detected
BCF (algae)
88
98
38- soil characteristics: sand 80%, silt 12%, clay 8%, organic matter 1.8%, pH
5.8- 2 fish used in each test, weight of fish: 15-25 g- fish fed in experiment A and B, no feeding in experiment C- test system with constant air supply, temperature: 25 +/- 2°C(2) valid with restrictions
Test design not equivalent to current standard methods;
isolated results, however, are considered valid.
Critical study for SIDS endpoint(23)

ADDITIONAL REMARKSUNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

4.1

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

ACUTE/PROLONGED TOXICITY TO FISH

Type
Species
Exposure period
Unit
LC0
Limit test
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:

semistatic
Brachydanio rerio (Fish, fresh water)
96 hour(s)mg/l>= .57

Method:Result:

Test condition
Reliability

:
:

Flag
20.10.2003:

Limit-test with saturated TS solution.
The test substance was crushed with a pestle, to accelerate
the adjustment of the TS concentration, 5 mg of the test
substance was added to 1 litre of water, the resulting
suspension was stirred on a magnetic stirrer for 24 hours,
treated in an ultrasonic bath for 1 hour, and finallyfiltered to remove undissolved particels of the test
substance; analyt. monitoring: GC
LC0 related to effective test substance concentration
measured daily before and after 24 h exposure (water change every 24 h).
Nominal concentration of 1 mg/l was measured:Start of incubation: 0.93, 1.09, 1.19, and 1.10 mg/l
End of incubation: 0.44, 0.63, 0.68, and 0.51 mg/l
21.4-21.9 °C; pH 7.6-8.1; dissolved oxygen 8.4-9.7 mg/l(2) valid with restrictions
Guideline study; recovery of test substance at terminationof exposure < 80 %Critical study for SIDS endpoint
(34)

Type
Species
Exposure period
Unit
Limit test
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

semistatic
Salmo gairdneri (Fish, estuary, fresh water)
96 hour(s)Remark:Result:

Test condition:

Reliability:

test substance was not completely soluble at concentrations
in excess of 0.6 mg/l
BHT was not toxic at a concentration of 0.6 mg/l (thresholdof water solubility). The lowest concentration at which
deaths were observed was 5 mg/l at which 1 of 10 fish diedover a 96 h exposure period.
10 fish in each aquarium; fish not fed; aquaria were gently
aerated; renewal of test solution daily; temperature: 15°C;
pH 8.1; water hardness: 250 mg/l as CaCO3; dissolved oxygen:
10.4 mg/l; solubilizing agent: 0.1 and 0.5 ml acetone/l,
respectively; solvent controls(3) invalid
methodological deficiencies; applied vehicle concentrations

yesother: Directive 84/449/EEC, C.1 "Acute toxicity to fish" (1992)
1994
yesother TS: purity 99.8 %

no
other
1982
yes
other TS: >= 99%

UNEP PUBLICATIONS

57

OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
exceeded limits fixed in OECD and EU guidelines (0.1 ml/l)

28.11.2001

(35)

Type
Species
Exposure period
Unit
LC50
Method

:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:Reliability:

Oryzias latipes (Fish, fresh water)
48 hour(s)mg/l5other: Japanese Industrial Standard (JIS K 0102-1986-71) "Testing
methods for industrial waste water"
no datano datareported EC50 exceeding the water solubility of the test
substance; not stated whether a static or a semi static
system was used
(3) invalid
effect concentration (LC50) above water solubility of TS;
not stated wether monitoring of test substance was
performed during exposure

30.11.2001

(31)

Type
Species
Exposure period
Unit
Limit test
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

staticOryzias latipes (Fish, fresh water)
48 hour(s)mg/lResult:

Test condition:

Reliability:

LC50: 17.5 mg/l (10°C)
13.5 mg/l (20°C)
5.3 mg/l (30°C)
body length: 2 cm, b.w. 0.2 g
solvent: 10 % ether-DMSO solution(3) invalid
reported effect levels exceeding the water solubility of thetest substance; documentation insufficient for assessment(literature in Japanese, only few data given in English)

no dataotherno datano data

30.11.2001

58

(36)

Type
Species
Exposure period
Unit
Limit test
Analytical monitoring
Method

:
:
:
:
:
:
:

Year
GLP
Test substance:::

Result:Brachydanio rerio (Fish, fresh water)noother: proposal of the Federal Environment Agency: Letale Wirkung beimZebrabärbling Brachydanio rerio (LC0, LC50, LC100; 48-96h) (1982)
1982
nono data
the applied test substance concentration of 100 mg/l had nolethal effect on exposed fish within the test period of 96
hours

UNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test condition:

Reliability:

10 fish pro aquaria; length of fish: 30 mm; no feeding;temperature during the test: 18.0-20°C; pH 6.7-6.0;
dissolved oxygen during the test: 9.0-7.8 mg/l; solubilizing
agent: ethanol(3) invalid
reported NOEC significantly exceeding the water solubilityof the test substance; no analytical monitoring of test
substance during exposure

30.11.2001

(33)

Type
Species
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

RemarkReliability::

Lepomis macrochirus (Fish, fresh water)
96 hour(s)other: no data
no datano data
No behavioural stress observed with 1.7 mg BHT/l.
(4) not assignablesecondary literature

12.11.2001

(15)

Type
Species
Exposure period
Unit
Limit test
Analytical monitoring
Method

:
:
:
:
:
:
:

Year
GLP
Test substance:::

RemarkResult::Reliability:

staticLeuciscus idus (Fish, fresh water)noother: Bestimmung der akuten Wirkung von Stoffen auf Fische. Arbeitskreis"Fischtest" im Hauptausschuss "Detergentien" (15.10.73)
1976
norange finding test; direct weight
the applied test substance concentration of 1000 mg/l had nolethal effect on exposed fish within the test period of 72
hours(3) invalid
reported LC0 by far exceeding the water solubility of thetest substance; no analytical monitoring

30.11.2001

(37)

Type
Species
Exposure period
Unit
Limit test
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: no data
Carassius auratus (Fish, fresh water)

RemarkResult::Reliability:

Exposure period: not stated
BHT was not toxic to goldfish in saturated solution (0.4mg/l; no further information)(4) not assignable

no dataother: no data
no datano data

UNEP PUBLICATIONS

59

OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
secondary literature

26.11.2001
4.2

(38)

ACUTE TOXICITY TO AQUATIC INVERTEBRATESTypeSpecies
Exposure period
Unit
EC0
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

staticDaphnia magna (Crustacea)
48 hour(s)mg/l>= .17yesother: Directive 67/548/EEC, C.2 "Acute Toxicity for Daphnia" (1992)
1994
yesother TS: purity 99.8 %Method:Result:Reliability:

Flag
20.10.2003:

Limit-test with saturated TS solution;
The test substance was crushed with a pestle, to accelerate
the adjustment of the test concentration, 5 mg of the test
substance was added to 1 liter of Elendt medium, theresulting suspension was stirred on a magnetic stirrer for
24 hours, treated in an ultrasonic bath for 1 hour, andfinally filtered to remove undissolved particels of the test
substance; analytical monitoring: GC
EC0 related to geometric mean of the test substance concentration
measured at beginning of test and after 48 hours of exposure(2) valid with restrictions
Guideline study; recovery of test substance at terminationof exposure < 80 %Critical study for SIDS endpoint

Type
Species
Exposure period
Unit
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

staticDaphnia magna (Crustacea)
48

Remark:Result:

Test condition:

Reliability:

test substance was not completely soluble at concentrations
in excess of 0.6 mg/l
The lack of a dose-response relationship and the fact thatthe majority of D. magna were trapped at the surface
suggests that immobilisation was due to the physicalpresence of undissolved material rather than to a toxic
effect.
10 daphnids < 24h old per test vessel; temperature: 20°C; pH
8.1; water hardness: 220 mg/l as CaCO3; dissolved oxygen:
9.2 mg/l; solubilizing agent: 0.5 ml acetone/l; solvent
controls(3) invalid
methodological deficiencies; applied vehicle concentrations
significantly exceeded limits fixed in OECD and EU
guidelines (0.1 ml/l)

(39)

no
other
yes
other TS: >= 99%

30.11.2001

60

(35)

UNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type
Species
Exposure period
Unit
EC50
Analytical monitoring
Method

:
:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:Test condition:

Reliability:

20.10.2003
4.3

staticDaphnia pulex (Crustacea)
48 hour(s)
mg/l
1.44
no
other: ASTM E729-80: Standard practise for conducting acute toxicity tests
with fish, macroinvertebrates and amphibians (1980)
no dataother TS: purity > 96 %the authors report solubility problems at higher
concentrations in the bioassay
10 neonates (<24h old) in each test chamber, not fed during
the test; test solution: reconstituted hard water;
temperature: 17°C, solving agent: acetone (0.5 ml/l);
solvent controls(2) valid with restrictions
The result is above water solubility. Methodological deficiencies:- maximum vehicle concentration exceeded limits fixed in OECD and EU
guidelines (0.1 ml/l)- although solubilizing agent was used, solubility problems are reported(40)

TOXICITY TO AQUATIC PLANTS E.G. ALGAE

Species
Endpoint
Exposure period
Unit
EC50
EC8
Limit test
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Scenedesmus subspicatus (Algae)
other: biomass and growth rate
72 hour(s)
mg/l
> .4
= .4
yes
yes
other: Directive 67/548/EEC, C.3 "Algal inhibition test" (1992)
1994
yesother TS: purity 99.8 %Method:Remark:Result:Limit-test with saturated TS solution.
The test substance was crushed with a pestle, to accelerate
the adjustment of the test concentration, 5 mg of the test
substance was added to 1 litre of water, the resulting
suspension was stirred on a magnetic stirrer for 24 hours,
treated in an ultrasonic bath for 1 hour, and finallyfiltered to remove undissolved particles of the test
substance; analytical monitoring: GC
at a measured test concentration of 0.40 mg/l (= geometricmean of analytical values at start and end of the test)
there was a slightly lower cell density at the end of test
as compared to control (304000 and 358000 cells/ml,
respectively); on the other hand, the cell density
multiplied by a factor of 30 within 72 hours, which is much
more than required for fullfilling the quality criteria with respect to thegrowth in the control (>= factor 16).
EC50 is given as geometric mean of the measured test
substance concentration at the beginning and end of test
after 72 hours of exposure.

UNEP PUBLICATIONS

61

OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability:

Flag
20.10.2003:

Species
Endpoint
Exposure period
Unit
Limit test
Analytical monitoring
Method

:
:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:Result:

Test condition:

Reliability:

A NOEC could not be derived.
(2) valid with restrictions
Guideline study; recovery of test substance at terminationof exposure < 80 %Critical study for SIDS endpoint(41)
Selenastrum capricornutum (Algae)
growth rate
96 hour(s)mg/lnoother: acc. to Miller & Green (1978): The Selenastrum capricornutum(Prinz) algal assay bottle test. EPA-60/9-78-018
1982
yesother TS: >= 99%
test substance was not completely soluble at concentrations
in excess of 0.6 mg/l
BHT was not toxic up to the threshold of water solubility(0.6 mg/l). At concentrations in excess of 6.6 mg/l the cellconcentration on day 4 was less than 50% of the mean control
cell concentration. Due to the nature of data, thecalculation of an EC50 was impossible.
S. capricornutum - initial concentration: 5x10E3 cells/ml;
temperature: 24°C; constant illumination; solubilizing
agent: 0.5 ml acetone/l; solvent controls(3) invalid
methodological deficiencies; applied vehicle concentrations
significantly exceeded limits fixed in OECD and EU
guidelines (0.1 ml/l)

30.11.2001
4.4

62

(35)

TOXICITY TO MICROORGANISMS E.G. BACTERIA

TypeSpecies
Exposure period
Unit
EC0
EC50
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

aquatic
activated sludge
3 hour(s)mg/l= 1000> 10000
other: Directive 88/302/EEC, Part C (Test for respiratory rate) (1988)
2000
yesother TS: 99.9 %Method:Reliability:

Flag
20.10.2003:

The purpose of the study is to provide a rapid screening method to identify
substances which may adversely affect aerobic microbial treatment in
wastewater treatment plants.
The respiration rate of activated sludge (0.32 g/l suspended solids) was
measured at 0, 100, 1000, and 10000 mg/l BHT after an incubation of 3hours at 20.1-20.2 °C and pH 8.1 (control 10000 mg/l without sludge pH
7.4).
(1) valid without restriction
Guideline study
Critical study for SIDS endpoint(42)

UNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type
Species
Exposure period
Unit
EC0
Analytical monitoring
Method

:
:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:Reliability:

aquatic
Pseudomonas putida (Bacteria)
30 minute(s)
mg/l
500
no
other: Bewertung toxischer Wasserinhaltsstoffe aus ihrer Inhibitorwirkung
auf die Substratoxydation von Pseudomonas Stamm Berlin mit Hilfe
polarographischer Sauerstoffmessungen. Robra, K.H.: gwf
wasser/abwasser 117 (2), 80-86 (1976)1984
noother TS: 99.8 %2 g/l Emulgator W; stock solution was stirred for 22 h andfilteredoxygen consumption test(2) valid with restrictions
study well documented, meets generally accepted scientificprinciples

30.11.2001

(43)

Type
Species
Exposure period
Unit
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

aquatic
Pseudomonas fluorescens (Bacteria)

Method:Result:

Test condition
Reliability::

Log phase cells, pre-grown in Difco nutrient broth, used for
exposure; inoculum density: 4 x 10E9 cells/ml; three nominal
concentrations tested: 10, 25 and 50 mg/l; TS predissolvedin ethanol; solvent control performed; 0.1 ml of incubatedsuspension diluted in phosphate buffer; triplicate samples;
viable cells determined by the spread plate technique on
nutrient agar plates after incubation at 20°C for 36 h
No growth inhibition observed up to the highest nominal
concentration tested (50 mg/l) after 1 hour of incubation
incubation at 20°C; shaking at 120 rpm(2) valid with restrictions
test procedure in accordance with generally acceptedscientific principles

no dataother: test on growth inhibition
no dataother TS: 99%

27.11.2001

(44)

Type
Species
Exposure period
Unit
EC50
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

aquatic
Tetrahymena pyriformis (Protozoa)
24 hour(s)
mg/l
1.7
no
other: cell multiplication inhibition test
1985
no dataother TS: dibutylhydroxytoluene (BHT), analytical grade

Method:The test was carried out under sterile conditions; test

UNEP PUBLICATIONS

63

OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:

Test condition
Reliability::

20.10.2003

organisms were precultured at 30°C for 24 h. For preparation of the stock
solution high concentrations of test substances of poor solubility werepredissolved in DMSO. The stock solution was added providing a constantratio of 1.8 in 10 ml of 2% proteose peptone; inoculation with 0.2 ml T.
pyriformis; vehicle control was performed; number of cells counted
manually under a microscope (repeated 3 times) or with a coulter counter(model Zb; repeated twice)
Correlation coefficient between manual counting and coultercounter: 0.998
Incubation at 30 °C without agitation(2) valid with restrictions
Test procedure in accordance with generally accepted scientific standards
(45)

Type
Species
Exposure period
Unit
IC100
Analytical monitoring
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

aquatic
Staphylococcus aureus (Bacteria)
24 hour(s)
mg/l
150
no
other: growth inhibition test

Result:

BHT concentration (mg/l)
Growth inhibition(three strains) (%)

Test condition:

Reliability:nono data

50
23-42
75
56-76
100
93-97
150
100
three strains tested in Brain Heart Infusion, BHT
concentrations used: 0-150 mg/l, temperature: 37°C, static
test system, two replications, solubilizing agent: 95% ethyl
alcohol, measurement of turbidity, nominal concentrations(3) invalid
documentation insufficient for assessment

28.11.2001

(46)

4.5.1

CHRONIC TOXICITY TO FISH

4.5.2

CHRONIC TOXICITY TO AQUATIC INVERTEBRATES

64

Species
Endpoint
Exposure period
Unit
NOEC
LOEC
Analytical monitoring
Method

:
:
:
:
:
:
:
:

Year
GLP
Test substance:::

Daphnia magna (Crustacea)
reproduction rate
21 day(s)
mg/l
.07
.39
yes
other: OECD Guide-line 202, part 2 "Daphnia sp., Reproduction Test" draft
1993
1994
yes
other TS: purity 99.8 %UNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Method:Remark:Test condition:

Reliability:

Flag
20.10.2003:

Species
Endpoint
Exposure period
Unit
Analytical monitoring
Method

:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:Result:

Test condition:

Reliability:- semi-static test with 3 test substance concentration applied (0.1, 0.316
and 1 mg/l; nominal)- the test substance was crushed with a pestle, to accelerate theadjustment of the test concentration 1 mg/l (= limit of water solubility), 5 mgof the test substance was added to 1 litre of Elendt medium, the resulting
suspension was stirred on a magnetic stirrer for 24 hours, treated in an
ultrasonic bath for 1 hour, and finally filtered to remove undissolved
particles of the test substance- test medium renewed 3 times a week; analytical monitored by GC after 48
and 72 h of exposure
EC0 is based on the geometric mean of the measured test substanceconcentrations (at the start and after 48 h and 72 h of exposure at waterchange). The value is 0.068 mg/l (geometric mean of 0.29; 0.03; 0.24; 0.01
mg/l)
20.0-21.6° C; pH 7.8-8.4; dissolved oxygen: 9.2-11.7 mg/l;
irradiation: 7.5 µE/m3 x s; light/dark-cycle: 16/8 h(2) valid with restrictions
Guideline study; recovery of test substance at terminationof exposure < 80 %Critical study for SIDS endpoint(47)
Daphnia magna (Crustacea)
reproduction rate
21 day(s)mg/lyesother: OECD Guide-line 202, part 2 " Daphnia sp. , Reproduction Test
(1984)
1986
noother TS: 98% supplied by Merck
Preparation of the stock solution by direct weight of 20,000
mg BHT into 2 l water of a gravel pit. Stirring for 4 days,
filtration with a membrane filter, and analysis on BHT
concentration. In 1986, instability of BHT in water was notrealized. So an accumulation of unknown impurities as wellas oxidation products of BHT in unknown amounts are likely
by the excessive entry of the test substance.
reduction of reproduction rate
17% at 0.05 mg BHT/l;
20% at 0.16 mg BHT/l;
60% at 0.5 mg BHT/l;semi-static test system; temperature: 20°C; illumination:
400 lux(3) invalid
test results cannot be related to BHT, due to th 20,000 foldexcess entry of the test substance's solubility (see also
remark)30.11.2001

(48)

4.6.1

TOXICITY TO SEDIMENT DWELLING ORGANISMS

4.6.2

TOXICITY TO TERRESTRIAL PLANTS

Species:other terrestrial plant: Pisum sativum (3 cultivars)

UNEP PUBLICATIONS

65OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Endpoint
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

other: growth and morphological changes

Method:Remark:Result:

BHT emulsion prepared by dissolving 1, 5, 100 or 300 mg BHT
in a few drops of ethyl acetate and shaking with 4 ml waterplus 10 ul of Tween 80; 10 ul of emulsion was applied to 3
cultivars of pea seedlings at the 5th day of germination(2nd day after planting the seedlings), application to thepea plumule twice within 18 hours; plant elongation measured
after 6 days of further growth
the appplication of 5µg/BHT/plant corresponded to aconcentration of 100 mg/l to the plumule at two times.
Growth inhibition of 5 day old pea seedlings (3 cultivars):

other
no
other TS: BHT, not further specified

ug BHT/ growth increasment in [%] of control +/stand.der.
plant 'Laxton Progress No. 9' 'Alaska' 'Sugar Snap'___________________________________________________________
1500
34 +/- 9
16 +/- 2 15 +/- 4
500
65 +/- 10
22 +/- 2 23 +/- 6
25
78 +/- 11
30 +/- 7 57 +/- 14
584 +/- 13
103 +/- 12___________________________________________________________

Reliability:

At 1500 ug BHT/plant 2 or 3 adventitious fast-growing shoots
arose from the base of the hypotcotyl; 500 and 25 ug caused
swelling of the upper part of the hypocotyl(3) invalid
Unsuitable method; test designed for basic research not for
ecotoxicological screening

30.11.2001

66

(49)

Species
Endpoint
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

other terrestrial plant: Malus domestica Borg.
other: growth and morphological changes

Method:Remark:Result:

BHT emulsion prepared by dissolving 0.1-3 g of BHT in 2 mlof ethyl acetate which was poured into stirred 100 ml waterplus 1 ml of Tween 80; treatment of 2, 3.5 and 7 month oldseedlings in three ways: (1) two drops applied to the apex,(2) spraying the whole plant, (3) dipping the upper part(with 5 leaves) of the plants; new growth increment measured
after 2 months of further growth
the appplication of 5µg/BHT/plant corresponded to aconcentration of 100 mg/l to the plumule at two times.
Two drops of BHT (10 ug/l) applied 5 times to the apex,
almost completely inhibited growth of 2 month old seedlings,
spraying and dipping were less effective; four to five weeks
later all seedlings resumed growth; after application of 1
and 5 ug BHT/l newly-grown parts of the stem had longer

other
no
other TS: BHT, not further specified

UNEP PUBLICATIONSOECD SIDS
4. ECOTOXICITY

Reliability

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

internodes than those of the controls; after spray treatment
with 20 and 30 ug BHT/l one-third of exposed 3.5 month oldseedlings produced a bunch of new shoots (2-5) which arosefrom the top of the plant, the number of shoots per plant
was proportional to the applied BHT concentrations withhigher concentrations provoking a larger number of new
shoots(3) invalid
Unsuitable method; test designed for basic research not for
ecotoxicological screening

30.11.2001(49)

Species
Endpoint
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

other terrestrial plant: Lactuca sativa L.
other: growth and morphological changes

Method:Result:

Test condition
Reliability::

BHT dissolved in ethyl acetate was applied to filter-paper
discs in 6 concentrations (10-300 ug per dish); after
drying, discs were wetted with water and put into petri
dishes with 10 lettuce seeds; incubation for 36, 48 or 72
hours, when plant elongation and morphological changes wererecordedNone of the applied BHT concentrations caused any inhibitionof germination under light or dark conditions; after 3 daysof incubation primary roots and the hypocotyls of the BHT
treated plants showed reduced growth (by half in the lightand several times more in the dark as compared to controls);
light-grown plants were swollen and greenish-yellow incolour; BHT-grown plants did not resume growth for at least
3 weeks; after transplanting into soil, some of the treatedplants developed 1 or 2 additional growing points 3 weeks
later, which produced small, malformed leaves; some of theleaf blades split along the central vein into 2 separate
parts which continued independent growthincubation at 22° C in the light or in the dark(3) invalid
Unsuitable method; test designed for basic research not for
ecotoxicological screening

other
no
other TS: BHT, not further specified

30.11.2001

(49)

4.6.3

TOXICITY TO SOIL DWELLING ORGANISMS

4.6.4

TOX. TO OTHER NON MAMM. TERR. SPECIESSpecies
Endpoint
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

other avian: White leghorn chicks (Gallus domesticus)
weight42 day(s)
other
1985
no
other TS: Butylated hydroxytoluene

UNEP PUBLICATIONS

67OECD SIDS
4. ECOTOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Method:Result:Reliability:

20.10.2003- Chicks were provided with feed containing 0.1, 0.4 and 1.0 % BHT(corresponding to 8 x, 30 x and 80 x of the standard BHT concentration[0.013 %] in feed) beginning at 1st day of age and continuing for the next 6
weeks- Beginning from day 8 chicks were fed "3000 ppb aflatoxins" (= 2722 ppb
aflatoxin B1, 99 ppb aflatoxin B2, 550 ppb aflatoxin G1, 79 ppb aflatoxin
G2). Control feed contained 107 ppb aflatoxins including 12 ppb aflatoxin
B1, 12 ppb aflatoxin G1, 77 ppb aflatoxin G2.
- BHT was dissolved in ethanol before mixing with feed and the solvent
was allowed to evaporate- Chicks were weighed weekly- Feed efficiencies were calculated from total weight gain and total feed
consumed
The influence of BHT on aflatoxin toxicity has been examined in a feeding
study.
0.1 % BHT in the diet (8x the standard BHT concentration of 0.013 % infeed) had no significant inhibiting effect on weight gain, whereas 0.4 % (30x the standard BHT concentration in feed) resulted in a temporary
depression (36 %) of weight gain from day 1 until 3 weeks; from week 3 toweek 6 the weight gain was 98 % of control and at the end of BHT
treatment (6 weeks) the total weight gain amounted to 78 % of control.
Both BHT dosages improved body weight gain and feed efficiency ofchicken treated with 3000 ppb aflatoxin in feed.
Feed containing 1 % BHT (80 x of normal concentration in feed) was only
fed until chicks were 3 weeks of age and resulted in 53 % inhibition ofweight gain.
(2) valid with restrictions
Basic data given(50)

Species
Endpoint
Exposure period
Unit
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

Coturnix coturnix japonica (avian)weight17 day(s)Remark:Reliability:

BHT was fed to 6 day old Japanese quail (Coturnix coturnix
japonica) for 17 days. The mean BHT intake was 1,100
mg/kg/day. At the earlier period of administration, BHT
reduced the body weight gains and food intakes, and increased the degreeof feather shedding. Results also suggest that there are differences in themetabolism of BHT between avians and mammals.
(4) not assignable
original reference not available(51)

24.06.2003

1981

4.7

BIOLOGICAL EFFECTS MONITORING

4.8

BIOTRANSFORMATION AND KINETICS

4.9

ADDITIONAL REMARKS

68

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY
5.0

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

TOXICOKINETICS, METABOLISM AND DISTRIBUTION

Remark:26.03.2003
5.1.1

BHT is readily absorbed through the gastrointestinal tract and, to a small
extent, through the intact skin. After long-term feeding of BHT-containing
diets the compound is accumulated especially in adipose tissue, while
lower levels are found in the liver, with elimination half-lives of 7 - 10 days
for both organs on cessation of treatment. In rats and probably also inhumans, an enterohepatic circulation takes place, particularly for the
metabolite BHT acid and its glucuronide. BHT is excreted primarily in theurine and, to a smaller extent, in the faeces. After a single oral application
to rats 80 - 90% of the dose were found in the urine within four days, mostof it within 24 hours. Rabbits excreted approximately 54% within four days
and humans 66% within 11 days in urine. Several metabolic pathways andmetabolites have been identified. BHT is activated by a cytochrome P450
dependent metabolic reaction in the liver.
(11)

ACUTE ORAL TOXICITY

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
> 2930 mg/kg bw
rat
Sprague-Dawley
male/female

Remark:Reliability
Flag
19.11.2001::

NUMBER OF ANIMALS: 5/dose/sex
MORTALITY: 0/10 (2150 mg/kg); 1/5 (f)/0/5 (2510 mg/kg) deathoccurred 5th day after application; 0/10 (2930 mg/kg)
CLINICAL SIGNS: no
BODY WEIGHT: no effectGROSS EXAMINATION: no effect
LD50 calculated according to the Bliss' method and that of
Litchfield & Wilcoxon(1) valid without restriction
Critical study for SIDS endpoint

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: an aqueous dispersion at 10% (W/V) of gum Arabic
OECD Guide-line 401 "Acute Oral Toxicity"
1988
yesother TS: Rhodianox BHT AP5 (white crystals, crystallization point: 69.4;
heavy metals: not detectable); min. 99.5 % BHT

(52)
LD50
> 10000 mg/kg bw
rat
Wistarmale/female
other: propyleneglycol
other: 1 dose level; 14 days observation period
1978
noother TS: Vulkanox KB (min. 99.5%)

UNEP PUBLICATIONS

69

OECD SIDS
5. TOXICITY

70

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Reliability:

Flag
19.11.2001

:

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 1700 mg/kg bw
rat

RemarkTest substance
19.11.2001::

male rats; no information on purity of TS
purified commercial product; 20 % solution (w/v) in corn oil

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 1970 mg/kg bw
rat

RemarkTest substance
19.11.2001::

male rats; no information on purity of TS
purified commercial product; 20 % solution (w/v) in corn oil

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

NUMBER OF ANIMALS: 10/dose/sex
APPLICATION: single dose of 30 ml per kg bw of 33% (w/v)
suspension in propylenglycol equivalent to 10 g BHT per kg
bw
MORTALITY: 0/20 (10 g/kg)
CLINICAL SIGNS: no
BODY WEIGHT: no data
GROSS EXAMINATION: no effect(2) valid with restrictions
Relatively high volume (30 ml per kg bw) administered
Critical study for SIDS endpoint(53) (54)other: Acute Oral Toxicity
1955
no dataother TS(55)

other: Acute Oral Toxicity
1955
no dataother TS(55)
LD50
= 2450 mg/kg bw
ratother: Acute Oral Toxicity
1959
no datano data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark19.11.2001

:

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 1906 mg/kg bw
rat

Remark19.11.2001:

male rats; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 2255 mg/kg bw
rat

Remark19.11.2001:

female rats; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 890 mg/kg bw
rat

Remark:male and female rats, unspecified
LD50 value converted from previous 0.89 g/kg.
Above value was most likely cited from 1969 alien reportrather than self-generated. No information on purity of TS.

male and female rats; no information on purity of TS(56)

other: Acute Oral Toxicity
1976
no datano data(57)

other: Acute Oral Toxicity
1976
no datano data(57)

other: Acute Oral Toxicity
1977
no datano data

19.11.2001
Type
Value
Species
StrainSex
Number of animals

(58)
:
:
:
:
:
:

LD50
= 2250 mg/kg bw
rat

UNEP PUBLICATIONS

71

OECD SIDS
5. TOXICITY

72

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:

other: Acute Oral Toxicity
1973
no datano data

Remark19.11.2001:

sex not specified; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 5800 mg/kg bw
rat

Remark19.11.2001:

sex not specified; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD100
= 3500 mg/kg bw
rat

Remark19.11.2001:

male and female rats; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 2000 mg/kg bw
mouseRemark19.11.2001:

sex not specified; no information on purity of TS

Type
Value

:
:

(59)

other: Acute Oral Toxicity
1976
no datano data(60)

other: Acute Oral Toxicity
1959
no datano data(56)

other: Acute Oral Toxicity
1959
no datano data(56)
LD50= 1800 mg/kg bw

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

mouse

Remark19.11.2001:

sex not specified; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 1040 mg/kg bw
mouseRemark:Test substance
19.11.2001:

male mice; observation period: 10 days; no information on
purity of TS
vehicle: 1 - 4 % solution in cottonseed oil

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD100
= 2500 mg/kg bw
mouseRemark19.11.2001:

male and female mice; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: Acute Oral Toxicity
1973
no datano data(59)

other: Acute Oral Toxicity
1949
no dataother TS(61)

other: Acute Oral Toxicity
1959
no datano data(56)
LD50= 3200 mg/kg bw
rabbitother: Acute Oral Toxicity
1973
no datano data

UNEP PUBLICATIONS

73

OECD SIDS
5. TOXICITY

74

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark19.11.2001:

sex not specified; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: "approx. lethal dose"
2100 - 3200 mg/kg bw
rabbitRemark:Test substance
19.11.2001:

male and female rabbits; observation period: 4 weeks; noinformation on purity of TS
purified commercial product; 20 % solution (w/v) in corn oil

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: "approx. lethal dose"
940 - 2100 mg/kg bw
cat

RemarkTest substance
19.11.2001::

male and female cats; no information on purity of TS
purified commercial product; 20 % solution (w/v) in corn oil

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: "approx. lethal dose"= 10700 mg/kg bw
guinea pig

RemarkTest substance
19.11.2001::

male and female guinea pigs; no information on purity of TS
purified commercial product; 20 % solution (w/v) in corn oil

Type
Value
Species
Strain

:
:
:
:

(59)

other: Acute Oral Toxicity
1955
no dataother TS(55)

other: Acute Oral Toxicity
1955
no dataother TS(55)

other: Acute Oral Toxicity
1955
no dataother TS(55)
LD50
= 2820 mg/kg bw
hamster

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Sex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

other: Acute Oral Toxicity
1973
no datano data

Remark19.11.2001:

sex not specified; no information on purity of TS
(59)

5.1.2

ACUTE INHALATION TOXICITY

5.1.3

ACUTE DERMAL TOXICITY

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

LD50
> 2000 mg/kg bw
rat
Sprague-Dawley
male/female

Remark:Reliability
Flag
26.03.2003::

NUMBER OF ANIMALS: 5/dose/sex
MORTALITY: 0/10 (2000 mg/kg bw)
CLINICAL SIGNS: no
LOCAL EFFECTS: no
BODY WEIGHT: no effect
LD50 calculated according to the Bliss' method and that of
Litchfield & Wilcoxon(1) valid without restriction
Critical study for SIDS endpoint

5.1.4

other: an aqueous dispersion at 10% (W/V) of gum Arabic
OECD Guide-line 402 "Acute dermal Toxicity"
1988
yesother TS: Rhodianox BHT AP5 (white crystals, crystallization point: 69.4;
heavy metals: not detectable); min. 99.5 % BHT(62)

ACUTE TOXICITY, OTHER ROUTES

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
Route of admin.
Exposure time
Method
Year
GLP

:
:
:
:
:
:
:
:
:
:
:
:
:

LD50
138 - 1739 mg/kg bw
mouse

i.p.
other: Acute i.p. Toxicity
1981
no data

UNEP PUBLICATIONS

75

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test substance:other TS: vehicle: corn oil

Remark:In this study the time course and extent of lung injury andrepair was examined in 4 mouse strains. The data indicatethat all strains develop similar levels of injury atequivalent doses. The extent of lung damage however does notcorrespond with the lethal dose. No information on purity ofTS.

19.11.2001(63) (64)

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
Route of admin.
Exposure time
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 3550 mg/kg bw
mouseother: Acute i.p. Toxicity
1980
no datano data

Remark19.11.2001:

male mice; mouse strain: ddY; no information on purity of TS

Type
Value
Species
StrainSex
Number of animals
VehicleDoses
Route of admin.
Exposure time
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark

:

i.p.

(65)
LD50
= 400 mg/kg bw
mouse

i.p.
other: Acute i.p. Toxicity
1974
no dataother TS: vehicle: corn oil
male mice; mouse strain: Swiss-Webster; observation period:
9 days; no information on purity of TS

19.11.2001
Type
Value
Species
StrainSex
Number of animals
VehicleDoses
Route of admin.
Exposure time
Method
Year
GLP

76

(66)
:
:
:
:
:
:
:
:
:
:
:
:
:

LD50
= 8000 mg/kg bw
laboratory animali.p.
other: Acute i.p. Toxicity
1952
no data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test substance:no data

Remark19.11.2001:

observation period: 1 month; no information on purity of TS

5.2.1

(67)

SKIN IRRITATION

Species
Concentration
Exposure
Exposure time
Number of animals
VehiclePDII
ResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:rabbitslightly irritatingother: Skin Irritation1949
no dataother TS: vehicle: ether
Non-occlusive application of 420 mg 2,6-di-tert-butyl-pcresol/kg bw (unspecified contact period; no information on
purity of TS) to the shaved back of the rabbit.
Result: some erythema and superficial sloughing but nosevere tanning effect.
Application of 3300 mg solid 2,6-di-tert-butyl-p-cresol/kg
bw on gauze, held in place by a flexible wire screen(according to Draize et al. (1944)), did not cause any skinirritation or apparent systemic effects; contact period: 24hours.

19.11.2001(61)

Species
Concentration
Exposure
Exposure time
Number of animals
VehiclePDII
ResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

rabbitundiluted
Semiocclusive
24 hour(s)6Result:

Test condition:

After semi-occlusive application of test substance to theintact and scarified skin, respectively, a very slightirritation could be noted: Intact skin: erythema 3/6 (24 h);
2/6 (72 h), edema 1/6 (24 h); 0/6 (72 h); abraded skin:erythema 1/6 (24 h); 0/6 (72 h), edema 1/6 (24 h); 0/6 (72h)
TEST ANIMALS: Strain: New Zealand White (age: young adult;
sex: not specified)
ADMINISTRATION/EXPOSURE- Area of exposure: application of test substance to theintact and scarified skin
- Total volume applied: 500 mg

slightly irritatingother: Patch Test
1976
no dataother TS: Compound WTR 15 Nonox TBC (corresponding to Vulkanox KB)

UNEP PUBLICATIONS

77

OECD SIDS
5. TOXICITY

Reliability
Flag
08.09.2001
5.2.2

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004::

EXAMINATIONS- Scoring system: according to Draize (not specified)- Examination time points: 24 and 72 hours after application(2) valid with restrictions
Critical study for SIDS endpoint(53) (68)EYE IRRITATION

Species
Concentration
Dose
Exposure time
Comment
Number of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:Test condition:

Reliability
Flag
08.09.2001::

Species
Concentration
Dose
Exposure time
Comment
Number of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:rabbitundiluted
100 other: mg
6slightly irritatingother: Draize test
1976
no dataother TS: Compound WTR 15 Nonox TBC (corresponding to Vulkanox KB)
6 of 6 rabbits showed slight conjunctivitis after 24 hours.
Symptoms were completely reversible after 72 hours. No
further data available.
TEST ANIMALS: Strain: New Zealand White (age: young adult;
sex: not specified)
APPLICATION: eyes were not rinsed
EXAMINATIONS- Scoring system: according to Draize (not specified)- Observation period: 24, 48 and 72 hours after application(2) valid with restrictions
Critical study for SIDS endpoint(53) (68)
rabbitnot irritatingother: Eye Irritation1949
no dataother TS: 40 % solution in diallyl phthalate
Application of 30 ul test substance directly to the cornea
did not cause any corneal injury. The authors suggested,that this result might indicate that 2,6-di-tert-butyl-pcresol had a much greater affinity for the relatively blandmedium in which it was dissolved rather than for the corneal
or conjunctival membranes. No information on purity of TS

19.11.2001

78

(61)

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY
5.3

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

SENSITIZATION

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Draize Testguinea pig

Remark:Test substance
19.11.2001:

Induction:
10 intracutaneous injections of test substance; frequency oftreatment: 3 days per week; first treatment: 0.04 mg in
0.05 ml vehicle, second to 10th treatment: 0.08 mg in 0.1 ml
vehicle;challenge:
2 weeks after last induction treatment 0.04 mg were
injected; 5 animals were tested.
No information on purity of TS.
purified commercial product; vehicle: 10 % aqueous ethanol

TypeSpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Remark:For a 2-year period (September 1987 to December 1989) 1336
consecutive eczema patients were patch tested. The patches
had been left on the skin for 2 days and readings had been
performed after 2 days, 3 days, and 1 week. All patch tests
were negative. (There was a frequency of 27 % withoccupational or doubtful occupational relevance, which
compares to other studies.)
No information on purity of TS.

not sensitizing
other: Skin Sensitization Test
1955
no dataother TS(55)

not sensitizing
other: Finn Chamber Test
1987
no dataother TS: 2 % in petrolatum

19.11.2001

(69)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Testlaboratory animal

Remark:Neat 2,6-di-tert-butyl-p-cresol was applied; no further
data available. No information on purity of TS.

not sensitizing
other: Skin Sensitization Test
1952
no datano data

UNEP PUBLICATIONS

79

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

19.11.2001

(67)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Remark:>= 15 individuals were tested with 100 % 2,6-di-tert-butylp-cresol, applied to the skin of the back for 48 hours;
challenging was after 14 days, provoking a moderatesensitizing effect (dull-red discolouration with edema,slight maceration, and possibly small petechiae). No
information on purity of TS.

sensitizing
other: Skin Sensitization Test
1952
no datano data

19.11.2001

(67)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Remark

:20 workers in the aerospace industry handling dielectric
fluids for electrodischarge machining and having developed
irritant contact dermatitis were patch tested with 2 %2,6-di-tert-butyl-p-cresol in petrolatum. The dielectric
fluids contained 0.2 % 2,6-di-tert-butyl-p-cresol as anadditive. All patch tests were negative.

not sensitizing
other: Skin Sensitization Test
1993
no datano data

22.04.1994

(70)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Result:11/11454 patients showed a positive reaction with BHT;
51/11454 showed a questionable/irritative response. No
information on purity of TS.

petrolatum

other TS: 2 % in petrolatum

19.11.2001

80

(71)

Type
Species::

Patch-Test
human

Result:With the standard procedure 1/155 contact dermatitis

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
patients reacted positive with BHT; patch-testing withremoval at 3 days and reading 3 h later 2/151 patients werepositive. No information on purity of TS.

19.11.2001

(72)

Type
Species::

Patch-Test
human

Result:One patient with food dermatitis was negative with BHT. No
information on purity of TS.

19.11.2001

(73)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Result:One case report described a contact dermatitis patient withpositive patch test results for tert. butylhydroquinone,BHT
and BHA. No information on purity of TS.

petrolatum

other TS: 1 % in petrolatum

19.11.2001

(74)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Patch-Test
human

Result:1/17 contact dermatitis patients (metalworkers) has apositive reaction with BHT. No information on purity of TS.

petrolatum

other TS: 1 % in petrolatum

19.11.2001

(75)

Type
SpeciesNumber of animals
VehicleResultClassification
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: Allergic shock test
guinea pig

Remark:Test substance:

0.08 mg 2,6-di-tert-butyl-p-cresol in 0.1 ml vehicle were
injected i.p.; 2 weeks later a "shock dose" of 0.16 mg in
0.2 ml vehicle was administered;result: no signs of anaphylaxis. No information on purity ofTS.
purified commercial product; vehicle: 10 % aqueous ethanol

not sensitizing
other: Sensitization Test
1955
no dataother TS

UNEP PUBLICATIONS

81

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

19.11.2001

(55)

5.4

82

REPEATED DOSE TOXICITY

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Result:

Test condition:

Reliability

:

rat
male
Sprague-Dawleyoral feed
40 daysdaily
no data specified
0.58, 0.69, 0.82, 1.00, 1.20, or 1.44 % in diet (ca. 436, 526, 663, 713, 774,
and 874 mg/kg bw day, based on body weight on day 10)
yes, concurrent no treatment
other: Subacute Oral Toxicity
1978
no datano data
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:- Mortality and time to death: no deaths in control and
0.58% BHT group; according to graphical plot of death rates
dose-related mortality at levels >=0.69%; 21/50 animals diedin the five highest dose groups during a period of 9 to 37
days; LD50 (40 days): 760 (669-864; 95% confidence limits)
mg/kg bw day- Clinical signs: mild diarrhea, rough hair, redish urine inrats fed BHT- Body weight gain: dose-dependent decrease (no data)- Food/water consumption: dose-dependent decrease (no data)- Organ weights: relative liver weight significantly
increased in BHT-treated rats (1.2 to 1.5 fold of control);no marked changes in the weights of kidney and heart; spleen
weight decreased in the same proportion as body weight (nofurther data)- Prothrombin index: A dose-dependent decrease in theprothrombin index was found in the survivors of all dosegroups (approximative indices as derived from figure: 35,
30, 25, 18, 20, 15% of control at 436, 526, 663, 713, 774,
and 874 mg/kg bw day).
- Haemorrhages: In dead animals a massive haemorrhage wasfound in the pleural and peritoneal cavities. Furthermore,haemorrhage occurred in other organs such as epididimys,
testis and pancreas of the survivors.
TEST ORGANISMS- Age: 4 weeks- Number of animals: 10 per group
CLINICAL OBSERVATIONS:The outward signs of intoxication, food and water
consumption, and body weight were recorded.
ORGANS EXAMINED:
Under ether anaesthesia, citrated plasma was collected fromthe inferior vena cava of the 40-day survivors. Prothrombin
time was estimated by the one-stage "Quick" method.
Exterior and interior haemorrhages were recorded in all
surviving animals. After sacrifice, the weights of theliver, right kidney, heart and spleen were determined.
(2) valid with restrictions

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Flag
29.10.2001:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:Reliability
Flag
19.11.2001

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
NOAEL
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark:Critical study for SIDS endpoint
(76)
rat
male
Sprague-Dawleyoral feed
1 or 4 weeks
daily
no data specified
0.0085 - 0.5 % in diet (1 week: ca. 7.54 - 529 mg/kg bw day; 4 weeks: ca.
6.73 - 326 mg/kg bw day)yes, concurrent no treatment
other: Subacute Oral Toxicity
1978
no datano data
After 1 week, a significant decrease in the prothrombin
index was observed at doses as low as 0.017 % (14.7 mg/kg bw
day) in diet, while at 4 weeks only the highest dose group(0.5 % in diet; ca. 326 mg/kg bw day) showed a decrease inthis index. Hence, the effect was transient.
The decline in prothrombin indices measured after 1 week
(100, 90.5, 85.9, 82.2, 78.4, 80.9, 84.4 and 73.8% at 0,
7.54, 14.7, 34.1, 62.5, 129, 227 and 529 mg/kg bw day,respectively) is not markedly dose-related and only fiveanimals were used per group.
(2) valid with restrictions
Critical study for SIDS endpoint
(77)
rat
male
Fischer 344
oral feed
76 weeksdaily
none
100, 300, 1000, 3000 and 6000 ppm
and 450 mg/kg bw day)yes, concurrent no treatment
1000 ppm
other: see remark field1990
no dataother TS: purity: > 99 %(ca. 7.5, 23, 75, 225

The study was not designed as definitive chronic bioassay.
21 rats /dose and 36 control rats; the diets were prepared
every 4 weeks and stored at 4°C until use (no analytical
data available); interim kill at 12, 36 and 48 weeks of 4
randomly selected animals; observations of pathology: To
demonstrate a deficiency in iron storage in cells of alteredhepatocellular foci, rats were iron-loaded with sc
injections of 12.5 mg elemental iron/100 g body weight in
the inguinal regions, alternating sides 3 times/week for 2
weeks prior to killing. Complete autopsies livers were

UNEP PUBLICATIONS

83

OECD SIDS
5. TOXICITY

84

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:performed on all animals. At autopsy, livers were weighedand slices from each lobe were taken and fixed in 10%
neutral buffered formalin. Sections were stanied withhaematoxylin and eosin and tested for iron to determine thepresence of iron storage-deficient lesions. Tumours andlesions other organs were submitted for histology.
All scheduled rats survived for up to 76 weeks
6000 ppm:
BODY WEIGHT: decreased
LIVER WEIGHT: increased
HISTOPATHOLOGICAL EXAMINATION (liver): no altered foci by 36
weeks; slightly, but not significantly altered foci at 48
and 76 weeks; after 76 weeks slightly increased incidence ofhepatic adenomas (33 %)3000 ppm:
BODY WEIGHT: decreased
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no altered foci by 36
weeks; slightly, but not significantly altered foci at 48
and 76 weeks
1000 ppm:
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no altered foci by 36
weeks; slightly, but not significantly altered foci at 48
and 76 weeks
300 ppm:
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no altered foci by 36
weeks; slightly, but not significantly altered foci at 48
and 76 weeks
100 ppm:
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no altered foci by 36
weeks; slightly, but not significantly altered foci at 48
and 76 weeks(2) valid with restrictions
Critical study for SIDS endpoint
(78)
rat
male
Fischer 344
oral feed
110 weeks
daily
none
12000 ppm (ca. 900 mg/kg bw day)yes, concurrent no treatment
other: Chronic Oral Toxicity
1990
no dataother TS: purity: > 99 %Mild epithelial hyperplasia of the forestomach was found in
61 % of the treated animals, compared to 48 % of the control
rats.
The incidence of altered hepatocellular foci was slightly

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:
:

Frequency of treatm.
Post exposure period
Doses

:
:
:

Control group
NOAEL
Method

:
:
:

Year
GLP
Test substance:::

Remark:decreased compared to control; the incidence of hepatocellular neoplasms was lower than in the control group; thetumour incidence was not increased.
Survival did not differ between dose and control group; bodyweight gain was reduced significantly compared to control;
liver weight was decreased significantly without affecting
the liver/body weight ratio;
27 rats/dose and control group.
(2) valid with restrictions
Critical study for SIDS endpoint(78)
ratmale/female
Wistaroral feedmale: 14 weeks (P); 141-144 weeks (F1)female: 20 weeks (P); 141-144 weeks (F1)
daily
non
nominal: 0, 25 100 and 500 mg/kg bw day (P); 0, 25, 100 and 250 mg/kg
bw day (F1)yes, concurrent no treatment
25 mg/kg bw
other: Two generation carcinogenicity study; the F1 generation being
dosed for their entire lifespan (for further details see remark field and also
chapter 5.8)
1986
no dataother TS: purity > 99.5 %ADMINISTRATION OF BHT: The BHT was mixed into asemi-synthetic powdered diet in concentrations adjusted
according to food consumption. Diet was prepared every
second week. the stability of BHT in the diet was examined
four times during each of the feeding periods for the F0 andF1 generations. The actual levels of BHT in the prepared
diets were a few percent less than the added amounts.
NUMBER OF ANIMALS (F1): Control: 100/sex; 25 mg/kg bw day: 80/sex;
100 mg/kg bw day: 80/sex; 250 mg/kg bw day: 100/sex
SERUM CHEMISTRY (only high dose F1, 20/sex):
glucoseblood urea nitrogen
free and total cholesterol
triglycerides
phospholipids
BLOOD ANALYSES (only high dose F1):
haematocrit
haemoglobin
red and white blood celldifferential white cell counts
PATHOLOGY (only F1): Specimens from the liver, kidneys,
heart, lungs, brain, spleen, pituitary gland, thyroid,
thymus(if any), pancreas, adrenals, testes, ovaries, seminal
gland, uterus, mesenteric and axillary lymph nodes, salivary
gland, gastro-intestinal tract (six levels), urinary
bladder, spinal cord, peripheral nerve, skeletal muscle,bone, skin, mammary gland, eye and Harderian gland were
fixed in 10 % neutral buffered formalin and embedded in

UNEP PUBLICATIONS

85

OECD SIDS
5. TOXICITY

86

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:Reliability
Flag
19.11.2001

:
:

Type
SpeciesSex:::paraffin, and sections were stained with haematoxylin andeosin for histological examination. Other appropriate
staining methods were used for selected specimens.
SURVIVAL in Controls: 16 males and 17 females
EFFECTIVE NUMBERS: animals that survived beyond wk 43, thetime when the first tumour appeared in the spleen of a malerat in the high-dose group
500 mg/kg bw day (P):BODY WEIGHT: decrease (m/f)
250 mg/kg bw day (F1):BODY WEIGHT: decrease (m: 21%; f:16%)
SURVIVAL: increase (m: 44 f: 39)
SERUM CHEMISTRY: decreased levels of triglyceride (f/m)
BLOOD ANALYSES: no effect (data not tabulated)
PATHOLOGY: increased number of liver adenomas in the males(18 animals with adenoma/99 (= "effective numbers")
100 mg/kg bw day (P):BODY WEIGHT: no effect described
100 mg/kg bw day (F1)BODY WEIGHT: decrease (m: 11%; f:10%)
SURVIVAL: increase (m: 34; f: 26)
PATHOLOGY: no signifcant effect
25 mg/kg bw day (P):BODY WEIGHT: no effect described
25 mg/kg bw day (F1):BODY WEIGHT: decrease (m: 7%; f:3% only at week 34)
SURVIVAL: (m: 44; f: 39)
PATHOLOGY: no significant effect(2) valid with restrictions
Critical study for SIDS endpoint
(79)
rat
male
Sprague-Dawleyoral feed
1, 2, 3, 4, 5, 6, or 7 days
daily
no1.2% in diet (mean daily intake: 500 mg/kg bw at day 1; >1000 mg/kg bw
day for 3-7 days;
average intake throughout feeding ca. 1000 mg/kg bw day
nono dataother TS: purity: from Tokyo Kasei Kogyo Co.
Rats given 1.2% BHT in the diet for 1, 2, 3, 4, 5, 6, or 7days (average intake about 1000 mg/kg bw day) showed asignificantly reduction in plasma concentration of blood
coagulation factors II, VII, IX and X in a time-dependent
fashion.
(2) valid with restrictions
Critical study for SIDS endpoint
(80)
rat
male

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:Reliability
Flag
19.11.2001

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
NOAEL
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:Sprague-Dawleyoral feed
3 weeksdailyno(i) 0, (ii) 1.2% BHT, (iii) 1.2% BHT + 0.0005% phylloqinone, (iv) 1.2% BHT+ 0.05% phylloqinone
yes, concurrent no treatment
no datano data
The study provided indirect evidence of the antagonistic
effect of BHT on vitamin K. The simultaneous administrationof phylloquinone (vitamin K1) had a preventing effect on
haemorrhages and PI reduction.
(2) valid with restrictions
Critical study for SIDS endpoint
(81)
rat
male
Wistargavage28 daysdaily
none
0, 25, 250 and 500 mg/kg bw day
yes, concurrent vehicle= 25 mg/kg bw
other: see remark field1986
yesother TS: purity: 99.9 %; vehicle: arachis oil
EXPERIMENTAL DESIGN: Twenty rats were randomly allocated to
one of four groups and were given a dose of 25, 250 of 500
mg
BHT/kg bw day or vehicle alone for 7 days. The rats in the 500 mg/kg bw
day group
initially received doses of 750 mg BHT/kg bw day for the first 3
days and a dose of 500 mg BHT/kg bw day for the remaining days.
After 7 days the rats were killed by cervical dislocation
andautopsied.
In the next phase of the experiment, groups of ten rats weretreated with 0, 25, 250 or 500 mg BHT/kg bw daily for 28 days
and were then killed and autopsied. Small samples of liverand epididymal adipose tissue were stored at -20°C and later
analysed for BHT by HPLC.
EXPERIMENTAL TECHNIQUES USED TO EXAMINE LIVER TOXICITY:
BIOCHEMICAL ASSAYS:
Microsomal protein
Glucose-6-phosphatase
Epoxide hydrolase
Total cytochrome P-450
Cytochrome b5
Ethoxyresorufin-O-deethylase

UNEP PUBLICATIONS

87

OECD SIDS
5. TOXICITY

Result88

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

BHT oxidase
IMMUNOCYTOCHEMISTRY: sections of liver from rats killed
after 28 days were stained immunocytochemically forcytochromes P-448 and P-450 using the three-layer PAP methodof Sternburger (Immunocytochemistry, 2nd Ed. Raven Press,
N.Y. (1979))
MICROSCOPIC EXAMINATION: samples of the 4 major lobes were
fixed in 10% neutral buffered formalin; sections were
stained with haematoxylin and eosin, with Van Giesons´sstain for collagen and with Gordon and Sweet´s method for
reticulin
500 mg/kg bw day:
BODY WEIGHT: weight loss reversed when dose was reduced (7days); marginally lower than that of the control group (28days)
LIVER WEIGHT: marked increase (7 or 28 days)
BHT CONTENT: very little (liver, 7 or 28 days); 227.4 mg/kg
wet weight (7 days), 168.4 mg/kg wet weight (28 days)
LIVER BIOCHEMISTRY: increase of proteins (7 or 28 days);decrease in glucose 6-phosphatase activity (7 or 28 days);increase in ethoxycoumarin-O-deethylase- and epoxidehydrolase activity (7 or 28 days)
HISTOPATHOLOGICAL EXAMINATION:
After 7 days:
Periportal region
hepatocyte necrosis
2/5
fibrosis
3/5
hepatocyte hypertrophy
3/5
hepatocyte hyperplasia
4/5
glycogen accumulation
4/5
After 28 days:
Periportal region
hepatocyte necrosis
6/10
fibrosis
5/10
bile-duct cell proliferation 4/10
hepatocyte hypertrophy
2/10
hepatocyte hyperplasia
3/10
pigment-laden macrophages 3/10
glycogen depletion
7/10
glycogen accumulation
0/10
IMMUNOCYTOCHEMISTRY: moderately -increased staining
intensity in the hypertrophied viable hepatocytes adjacentto the areas of damage
250 mg/kg bw day:
BODY WEIGHT: no effect (7 or 28 days)
LIVER WEIGHT: moderate increase (7 or 28 days)
BHT CONTENT: very little (liver, 7 or 28 days); 66.6 mg/kg
wet weight (7 days), 119.8 mg/kg wet weight (28 days)
LIVER BIOCHEMISTRY: increase of protein (28 days); decreasein glucose 6-phosphatase activity (28 days); increase inethoxycoumarin-O-deethylase- and epoxide hydrolase activity(7 or 28 days)
HISTOPATHOLOGICAL EXAMINATION: glycogen accumulation (7days: (4/5) 28 days: (8/10));IMMUNOCYTOCHEMISTRY: no effects
25 mg/kg bw day:
BODY WEIGHT: no effect (7 or 28 days)
LIVER WEIGHT: slight increase (7 or 28 days)
BHT CONTENT: very little (liver, 7 or 28 days); 11 mg/kg wetweight (7 days), 15.5 mg/kg wet weight (28 days)
LIVER BIOCHEMISTRY: no effects (7 or 28 days)

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:Test substance
Reliability
Flag
19.11.2001

:
:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:HISTOPATHOLOGICAL EXAMINATION: no effects
IMMUNOCYTOCHEMISTRY: no effect(1) valid without restriction
Critical study for SIDS endpoint(82)
ratmale/female
Fischer 344
dermal4 weeks
3 times/weeknone
240 mg/animal
females: 2286 mg/kg bw day)yes, concurrent vehicle
other: Subacute Dermal Toxicity
1986
no dataother TS

Except for a slight retardation of growth in males, no
substance-related symptoms were found;
10 rats/sex/dose and control group.
food additive grade; purity: > 98.0 %; vehicle: DMSO
(2) valid with restrictions
Critical study for SIDS endpoint(83)
rat
no datano dataoral unspecified
6 - 26 daysdaily
no data specified
see remark
no data specified
other: Subacute Oral Toxicity
1982
no datano data- liver disfunction and histopathologic changes (> 25 mg/kg
bw day, 7 days; liver enlargement, centrilobular necrosis,
glutathion depletion, increase in transaminases, enzyme
induction, increases in phospholipids and cholesterol even in serum);- kidney (500 mg/kg bw day, 6 days; changes in electrolyte
excretion);- thyroid gland (>= 25 mg/kg bw day, 26 days; hypertrophy,
microfolliculation, increase in iodine uptake).
No information on purity of TS; incompletely documented
study.

19.11.2001
Type
Species(males: 1967 mg/kg bw day;

(84)
:
:

rat

UNEP PUBLICATIONS

89

OECD SIDS
5. TOXICITY

90

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Sex
StrainRoute of admin.
Exposure period

:
:
:
:

Frequency of treatm.

:

Post exposure period
Doses

:
:

Control group
NOAEL
Method

:
:
:

Year
GLP
Test substance:::

Remark:Result:male/female
Wistarother: dietmale: 5 weeks (P); 4 weeks (F1), 6, 11, 16 and 22 months (F1); female: 8
weeks (P)daily (during the period of mating, food pots were removed when male andfemales were mated)nonominal: 0, 25, 100 and 500 mg/kg bw day (P); 0, 25, 100 and 250 mg/kg
bw day (F1)yes, concurrent no treatment
25 mg/kg bw
other: Two generation study with emphasis on hepatocellular changes in
F1 generation (for further details see remark field and also chapter 5.8)
1994
yesother TS: purity: 99.96%
EXPERIMENTAL TECHNIQUES USED TO EXAMINE LIVER TOXICITY:
BIOCHEMICAL ASSAYS:
Glucose 6-phosphatase
Epoxide hydrolase
Glutathione S-transferase
Total cytochrome P450
Ethoxyresorufin O-deethylase
Pentoxyresorufin O-depentylase
Total glutathione
Total, microsomal and cytosolic protein
IMMUNOCYTOCHEMISTRY: Slides were stained with a three layer
biothinylated streptavidin horseradish peroxidase method andthe following polyclonal primary antibodies:anti rat Cytochrome P450 1A subfamily
anti rat Cytochrome P450 2B subfamily
anti murine microsomal Epoxide Hydrolase
MICROSCOPIC EXAMINATION: light and electron microscopy wereused; cellular proliferation using the technique of pulse
labelling with osmotic pumps containing bromodeoxyuridine
was only assessed in the high dose F1-animals beginning with
4 weeks after weaning
MICROSCOPIC EXAMINATION OF THE THYRIOD: The diagnostic
criteria for hyperactivity are the presence of some or allof the following:
Reduction of the follicular size
Absence or reduction of colloid
Irregularities in the follicular outline
Hyperaemia Increase in number of follicular cells
ADMINISTRATION OF BHT: the amount of BHT incorporated
initially per unit weight of diet was calculated from thefood consumption measured during acclimatisation and fromnormal growth rate of this strain of rats; throughout
pregnancy and lactation no effort was made to adjust dietary
BHT content in line with body weight gain during this time
Results Field 1
500 mg/kg bw day (P, females, 20 gestation day):
BODY WEIGHT: no effect
LIVER WEIGHT: increase
HISTOPATHOLOGICAL EXAMINATION (liver): 4/5 animals showed
mild centrilobular enlargement and eosinophilia
LIVER BIOCHEMISTRY:IMMUNOCYTOCHEMISTRY in the liver: no effect

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
500 mg/kg bw day (foetuses):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: a trend towards an increase inglucose6-phosphatase; activity; results for cytochrome P450
and its isoenzymes have not been presented
IMMUNOCYTOCHEMISTRY in the liver: no effect
500 mg/kg bw day (male pups, 21 days post partum):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: decrease
LIVER WEIGHT: decrease
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: increase inentoxyresorufinO-depentylase; increase in total cytochrome
P450; increase in glutathione S-transferase- and epoxidehydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: no effect
250 mg/kg bw day (F1, males 4 weeks post weaning):
LIVER TO BODY RATIO: increase
BODY WEIGHT: decrease
LIVER WEIGHT: decrease
HISTOPATHOLOGICAL EXAMINATION (liver): no effect (incl. cellproliferation)
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase
inethoxyresorufin O-deethylase; increase in glutathione
S-transferase- and epoxide hydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: no effect
250 mg/kg bw day (F1, males 6 months post weaning):
LIVER TO BODY RATIO: increased
BODY WEIGHT: below that of controls
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): centrilobular
enlargement and eosinophilia (4/5); no cell proliferation
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inglutathione S-transferase- and epoxide hydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: no effect
250 mg/kg bw day (F1, males 11 months post weaning):
LIVER TO BODY RATIO: increase
BODY WEIGHT: decrease
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement andeosinophilia(10/10), single altered hepatic focus (2/10),
periportal induction of GGT (8/10), no cell proliferation;(kidneys): chronic progressive nephropathy; (thyroid):
hyperactivity (10/10); (adrenals): no effects
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase in total
cytochrom P450; increase in glutathione S-transferase- andepoxide hydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: focal phenotypic orproliferative changes (2/19)

UNEP PUBLICATIONS

91

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
250 mg/kg bw day (F1, males 16 months post weaning):
LIVER TO BODY RATIO: increase
BODY WEIGHT: decrease
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(12/13), periportal induction of GGT (13/13), no cellproliferation; (kidneys): chronic progressive nephropathy;(thyroid): hyperactivity (13/13); (adrenals): no effects
LIVER BIOCHEMISTRY:statistical significant difference inpentoxyresorufin O-depentylase activity; increase in total
cytochrom P450; increase in glutathione S-transferase- andepoxide hydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: focal phenotypic orproliferative changes (8/13)
TOTAL THYROXINE (T4): no effect
250 mg/kg bw day (F1, males 22 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: below that of controls
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement andeosinophilia(18/19), nodules ((6/19) periportal induction of
GGT (17/17), no cell proliferation (only one animal
examined); (kidneys): chronic progressive nephropathy;(thyroid): hyperactivity (13/13); (adrenals): no effects
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase in total
cytochrom P450; increase in glutathione S-transferase- andepoxide hydrolase activity
IMMUNOCYTOCHEMISTRY in the liver: focal phenotypic orproliferative changes (14/19)
TOTAL THYROXINE (T4): no effectResults Field 2
100 mg/kg bw day (P, females, 20. gestation day):
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
100 mg/kg bw day (foetuses):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: activity; results for cytochrome P450and
its isoenzymes have not been presented
100 mg/kg bw day (F1, male pups, 21 days post partum):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: increase in pentoxyresorufin
O-depentylase activity; increase in total cytochrome P450;
increase in epoxide hydrolase activity
100 mg/kg bw day (F1, males, 4 weeks post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: below that of control
LIVER WEIGHT: no effect

92

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inethoxyresorufin O-deethylase; increase in glutathione
S-transferase- and epoxide hydrolase activity
100 mg/kg bw day (F1, males 6 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: below that of controls
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): entrilobular
enlargement and eosinophilia (3/5)
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inglutathione S-transferase- and epoxide hydrolase activity
100 mg/kg (F1, males 11 months post weaning):
LIVER TO BODY RATIO: increased
BODY WEIGHT: below that of controls
LIVER WEIGHT: increased
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(6/8);single altered hepatic focus (2/10), periportal
induction of GGT (3/8); (kidneys): chronic progressive
nephropathy;(thyroid): hyperactivity (6/8); (adrenals): noeffects
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inglutathione S-transferase activity
100 mg/kg bw day (F1, males 16 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: below that of controls
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(0/9),periportal induction of GGT (8/8); (kidneys): chronic
progressive nephropathy; (thyroid): hyperactivity(7/9);(adrenals): no effects
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inglutathione S-transferase- and epoxide hydrolase activity
TOTAL THYROXINE (T4): no effect
100 mg/kg bw day (F1, males 22 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: below that of controls
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(4/11), periportal induction of GGT (7/11); (kidneys):chronic progressive nephropathy; (thyroid):
hyperactivity(9/11); (adrenals): no effects
LIVER BIOCHEMISTRY: statistical significant difference inpentoxyresorufin O-depentylase activity; increase inglutathione S-transferase activity
TOTAL THYROXINE (T4): no effectResults Field 3
25 mg/kg bw day (P, females, 20. gestation day):
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): 1/5 animals showed

UNEP PUBLICATIONS

93

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
mild centrilobular enlargement and eosinophilia
25 mg/kg bw day (foetuses):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: results for cytochrome P450 and its
isoenzymes have not been presented
25 mg/kg bw day (F1, male pups, 21 days post partum):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: increase in epoxide hydrolase activity
25 mg/kg bw day (F1, males, 4 weeks post weaning):
LIVER TO BODY RATIO:no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): no effect
LIVER BIOCHEMISTRY: no effects
25 mg/kg bw day (F1, males 6 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver): centrilobular
enlargement and eosinophilia (3/5)
LIVER BIOCHEMISTRY: increase in epoxide hydrolase activity
25 mg/kg bw day (F1, males 11 months post weaning):
LIVER TO BODY RATIO: increased
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(0/8),single altered hepatic focus (1/8), periportal
induction of GGT (1/8); (kidneys): chronic progressive
nephropathy; (thyroid): no effect; (adrenals): no effects
LIVER BIOCHEMISTRY: no effects
25 mg/kg bw day (F1, males 16 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(3/9),no periportal induction of GGT; (kidneys): chronic
progressive nephropathy; (thyroid): no effect; (adrenals):
no effects
LIVER BIOCHEMISTRY: increase in epoxide hydrolase
TOTAL THYROXINE (T4): no effect
25 mg/kg bw day (F1, males 22 months post weaning):
LIVER TO BODY RATIO: no effect
BODY WEIGHT: no effect
LIVER WEIGHT: no effect
HISTOPATHOLOGICAL EXAMINATION (liver, incl. histochemical
staining): centrilobular enlargement and eosinophilia(1/13);

94

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
NOAEL
MethodYear
GLP
Test substance

:
:
:
:
:
:

Result:no periportal induction of GGT; (kidneys): chronic
progressive nephropathy; (thyroid): no effect (adrenals): noeffects
LIVER BIOCHEMISTRY: no effects
TOTAL THYROXINE (T4): no effect
CONCLUSIONS: No BHT effect was seen in F0 generation
although the livers from lactating dams were much larger
than those from respective controls and showed morphologicalevidence of considerable metabolic activity. The
histological and biochemical changes seen in the F1generation were similar to those reported by other workers
on the hepatic effects of BHT and are consistent with theeffects of an inducer of cytochromes P450. The nodules andglucose 6-phosphatase deficient AHF observed at Time Point 7of this experiment were probably induced by BHT. No evidenceof thyroid increased activity as a result of BHT
administration was observed at a dose level of 25 mg/kg bodyweight/day BHT. Hyperactivity occurred at dose levels of 100
and 250 mg/kg body weight/day BHT. It appeared that BHT gavesome protection against the development of chronic
progressive nephrophathy (CPN), because CPN was observed in
all rats (incl. controls) at every time point, but thedisease was less severe in rats treated with 250 mg/kg bw day. No
adverse effect of BHT was observed in the adrenals.
(1) valid without restriction
Critical study for SIDS endpoint(85) (86)mouse
male/female
B6C3F1oral feed
10 weeksdaily
none
0.25, 0.5, 1, 2 and 4 % in diet(males: ca. 410, 820, 1640,
3480 and 6960 mg/kg bw day; females: ca. 438, 875, 1750, 4130 and 8260
mg/kg bw day)yes, concurrent no treatment
3480 - 4130 mg/kg bw
1988
no dataother TS: purity: 96%
All of the mice, except for one male in the 1 % group andone male and three female mice in the control group thatdied accidentally, survived the entire treatment period. The
average rates of body weight gain for the male and female
mice given a diet containing 4 % test substance were lessthan 90 % of those of the control group. Histologicalexamination of major visceral organs of mice that had been
given the diet containing 4 % test substance showed marked
starvation atrophy of the spleen, heart and kidneys. No
significant gross or histological changes were detected in
major visceral organs of mice that had been given a dietcontaining 2 % or lower concentrations of test substance, orin the mice of the control group.
On the basis of these results, the maximum tolerated dose(MTD) of 2,6-di-tert-butyl-p-cresol in the diet was

UNEP PUBLICATIONS

95

OECD SIDS
5. TOXICITY

Test substance

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

estimated to be 2 % for B6C3F1 mice of both sexes;
10 mice/sex/dose and 20 mice/sex/control group.
Dose-finding study for 104 week feeding study.
purity: 96 % (an actual level of almost 50 % of the initial
2,6-di-tert-butyl-p-cresol content was determined in thefeed pellets, the distribution of the test substance in thepellets seemed to be inhomogenous)

19.11.2001

(87)

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:Test substance

:

mouse
male/female
B6C3F1oral feed
104 weeks
daily
16 weeks
1 and 2 % in diet (males: 1640 and 3480 mg/kg bw day; females: 1750 and
4130 mg/kg bw day)yes, concurrent no treatment
1988
no dataother TS: purity: 96%
Body weight was dose-dependently decreased; food intake was
unaffected in males, treated females showed an increasedfood intake compared to control (maybe due to foodspillage); survival was increased in a dose-related mannerin male rats; males showed foci of cellular alteration inliver, the number of foci per male mouse also revealed adose-response relationship. In male mice nuclear pleomorphism of hepatocytes was noted in nontumourous areas.
Regarding mice with no tumour, the average absolute andrelative liver weights of male mice in each treatment groupwere significantly greater than those of control male mice.
2 % in diet were considered to be the maximum tolerated dosein this study;
50 mice/sex/dose and control group.
Results concerning carcinogenic effects are depicted inchapter 5.7.
purity: 96 % (an actual level of almost 50 % of the initial
2,6-di-tert-butyl-p-cresol content was determined in thefeed pellets, the distribution of the test substance in thepellets seemed to be inhomogenous)

22.11.2000

96

(87)

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
NOAEL
Method
Year

:
:
:
:

mouse
male
other: Slc:ddYoral feed
30 daysdaily
none
1.35, 1.75, 2.28, 2.96, 3.85 and 5.00 % in diet
3370, 4980 and 5470 mg/kg bw day)yes, concurrent no treatment< 1570 mg/kg bw
other: Subacute Oral Toxicity
1992

UNEP PUBLICATIONS(1570, 1980, 2630,

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

GLP
Test substance::no dataother TS: purity: > 99 %Remark:SourceReliability
Flag
26.03.2003:::

In all of the test substance-treated groups the prothrombin
index was significantly decreased to 73 - 78 % of thecontrol value. The kaolin-activated partial thromboplastin
time index was also decreased significantly in the 1.35,
1.75 and 5.00 % dose groups to ca. 60 - 70 % of the control
value. Increased relative organ weights of lung and liver.
Fatty and green-coloured liver (several cases in all
dose groups) and misshapen kidneys (at 5.00 %) wereobserved. The number of mice with nephrosis as judged bysome tubular lesions was 2, 3, 6, 8, 10 and 10 of the 10
mice in each of the 1.35 - 5.00 % dose groups. The ED50 for
toxic nephrosis for 1 month of administration was 2300 mg/kg
bw day. No haemorrhages or other pathological changes wereobserved in the lungs; the soft-wood chip bedding had probably a
protecting effect against lung haemorrhages;
no mortality occurred in any group; 10 mice/dose and control
group (held in cages with soft-wood chips as bedding).
Shell(2) valid with restrictions
Critical study for SIDS endpoint

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
NOAEL
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:Source

:

(88)
mouse
male
other: Slc:ddYoral feed
21 daysdaily
none
0.5, 1.0 and 2.0 % in diet (660, 1390 and 2860 mg/kg bw day)yes, concurrent no treatment= 660 mg/kg bw
other: Subacute Oral Toxicity
1992
no dataother TS: purity: > 99 %In the 0.5, 1.0 and 2.0 % groups, respectively one (day 7),one (day 6) and two (days 5 and 6) mice died.
Massive haemorrhages in the lungs, blood pooling or engorgement in theliver, heart, cranial cavity and epididymal
adipose tissue, and enlargement of the lungs and liver wereobserved in all of the dead mice.
Increased relative organ weights of lung and liver. Hepatic
2,6-di-tert-butyl-p-cresol (BC) concentrations of< 0.043, 0.991 and 1.787 ug/g wet weight, respectively weredetected in the 0.5, 1.0 and 2.0 % groups; BC-quinone
methide was not detected. Prothrombin index was reduced up to 60 % ofcontrol and kaolin-activatedpartial thromboplastin time index was reduced up to 42 % of control in 1 %and
2 % groups (both parameters statistically significantly reduced only in the
1.0 % group);
5 mice/dose and control group (held in stainless steel
wire-mesh bottomed cages).
Shell

UNEP PUBLICATIONS

97

OECD SIDS
5. TOXICITY

98

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
NOAEL
MethodYear
GLP
Test substance

:
:
:
:
:
:

Result:

Test condition:(2) valid with restrictions
Critical study for SIDS endpoint
(88)
mouse
male/female
CD-1
dermal4 weeks
3 times/weeknone
5, 10, 20 and 30 mg/animal (males: 145, 289, 578 and 867 mg/kg bw day;females: 208, 415, 830 and 1245 mg/kg bw day)yes, concurrent vehicle< 200 mg/kg bw
other: Subacute Dermal Toxicity
1986
no dataother TS: food additive grade; >98% purity
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:- Mortality and time to death: no deaths in control groups;
dose-dependent mortality rates: (i) males: 5 mg group 0/10;
10 mg group 1/10; 20 mg group 7/10; 30 mg group 8/10; (ii)females: 5 mg group 1/10; 10 mg group 9/10; 20 mg group
10/10; 30 mg group 10/10. Lethal effect more manifest infemale than in male mice.
- Clinical signs: dose dependent respiratory distress between days 4 and 8of the study with subsequent mortality, lethargy and weightloss after 2nd application in 20 and 30 mg groups, after 3rd
application also in 10 mg group (most females, one male) and
5 mg group (one female). At the lowest dose no toxic effects
were found in male mice.
- Gross pathology: At autopsy dead animals were found tohave congestion and enlargement of the lung with oozing offroth from the trachea. Other organs appeared normal in allgroups.
- Histopathology: Histologically, collapse of the alveoli
and dilatation of the alveolar ducts associated withdegeneration and necrosis of the type I alveolar epithelial
cells were evident. The type II alveolar epithelial cells
increased in number.
- Effects on skin: The treated skin showed epidermal
hyperplasia, occasionally associated with small ulcer
formation around hair follicles.
TEST ORGANISMS- Age: 8 weeks- Weight at study initiation: 34.6 g (males), 24.1 g(females)- Number of animals: 10 per sex and group
ADMINISTRATION / EXPOSURE- Vehicle: DMSO
- Total volume applied: 0.1 ml of DMSO solutions
CLINICAL OBSERVATIONS AND FREQUENCY:
Clinical signs, mortality
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC ANDMICROSCOPIC):- Macroscopic: lung and other organs (not specified)- Microscopic: lung
NOTE: Special study on pneumotoxicity

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Remark:SourceReliability
Flag
26.03.2003

:
:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:Test substance
Reliability
Flag
19.11.2001:::(2) valid with restrictions
Critical study for SIDS endpoint(83)
guinea pig
male
Hartley
oral feed
14 or 17 days
daily
none
0.125, 0.25, 0.5, 1.0 and 2.0 % in diet (85, 159, 235, 408 and 660 mg/kg
bw day)yes, concurrent no treatment
other: Subacute Oral Toxicity
1992
no dataother TS: purity: > 99 %Diarrhoea and rough hair were found in the 1.0 and 2.0 %groups from day 2 to 17. No haemorrhages in the lungs but asmall haemorrhage in the abdominal cavity was observed in asingle guinea pig at 1.0 %. The prothrombin index was
reduced at 0.5 % BHT and above (statistically significantly reduced in the
1.0 % group only) up to 77 % of control. The hepatic concentration of 2,6di-tert-butyl-p-cresol was increased dose-dependently, but its quinone
methide metabolite was not detected. Treated groups fed up to 0.25 % test
substance were dosed for 14 days, the others were treated for 17 days;
5 animals/dose and control group (14 days)
6 animals/dose and control group (17 days).
Shell(2) valid with restrictions
Critical study for SIDS endpoint(88)
hamstermaleother: Syrian golden
dermal4 weeks
3 times/weeknone
480 mg/animal (ca. 3097 mg/kg bw day)yes, concurrent vehicle
other: Subacute Dermal Toxicity
1986
no dataother TS
During the exposure period, no clinical abnormalities werenoted other than a slight retardation of growth. Autopsy
revealed no gross pathological lung lesions. There wereno histopathological changes;
10 animals/dose and control group.
food additive grade; purity: > 98 %; vehicle: DMSO
(2) valid with restrictions
Critical study for SIDS endpoint(83)

UNEP PUBLICATIONS

99

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:
:

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

Result:monkeymale/female
other: Macaca mulatta (Rhesus monkey)
gavage28 daysdaily
24 hours fasting before sacrifice
500 mg/kg bw per day (group I: infants, group II: juveniles); 50 mg/kg bw
per day (group III: juveniles)
yes, concurrent vehicle
other
1972
no
no data
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:- Mortality and time to death: no mortalities- Clinical signs: no abnormalities observed- Body weight gain: normal food consumption (no data)- Haematology/Urinalysis: no effects- blood: Biochemistry during exposure: no effects- liver: Biochemistry post-mortem studies: slightly modified enzyme activityof hepatic microsomes in 500 mg/kg bw day juveniles group (statisticallysignificant increase of nitroanisole demethylase activity by week 2 and 4
and decrease of glucose-6-phosphatase activity by week 4), not in 50
mg/kg bw day juveniles or 500 mg/kg bw day infants group; no effects onprotein content, total lipids, RNA, DNA or cytochrome P450 in all treatedgroups- Organ weights: no effect on relative liver weight in alltreated groups (in contrast to parallel experiments withbutylated hydroxyanisole (BHA))- Histopathology:
Indication of hepatocytomegaly and enlargement of hepatic
cell nucleoli in the treated juvenile groups.
500 mg/kg bw day groups of infants and juveniles: moderate
proliferation of hepatic endoplasmic reticulum, lipid(droplets) accumulation in cytoplasm probably due to use ofcorn oil (also observed in controls); nucleolar
fragmentation and presence of large intranuclear fibrils
observed in many of the hepatic nuclei.
500 mg/kg bw day group of infants: much less responsive than the mature
animals (This lower response of infants as compared to juveniles isprobably related to the lower activity of drug-metabolizing enzymes and isstrongly suggesting that metabolites were responsible for most of thechanges observed.)
50 mg/kg bw day group of juveniles: cytoplasmic changes less obvious; nonucleolar changes

Test condition

100:

Histopathological evaluation of all organs other than liverfrom either infant or juvenile animals showed no major
BHT-related pathological changes.
TEST ORGANISMS- Age: (i) 1-month old infant; (ii) immature (juvenile)monkeys- Weight at study initiation: (i) ca. 600 g; (ii): ca. 2.5
kg

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004- Number of animals: (i) 500 mg/kg bw day infants group: 3(control: 3); (ii) 500 mg/kg bw day juveniles group: 3 (vehicle
control: 2; control without vehicle: 3); (iii) 50 mg/kg bw day
juveniles group: 2 (vehicle control: 2)
ADMINISTRATION / EXPOSURE- Type of exposure: intragastric intubation- Vehicle: corn oil- Concentration in vehicle: 25% (w/v)

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

Remark:

CLINICAL OBSERVATIONS AND FREQUENCY:- Clinical signs: daily- Haematology: weekly determionation of complete blood count- Biochemistry: weekly determination of serum sodium andpotassium, bilirubin, cholesterol and glutamic-oxalacetictransaminase (SGOT). Post-mortem determination of following
liver parameters: protein, DNA, RNA. nitroanisoledemethylase, glucose-6-phosphatase, cytochrome P450, neutral
cellular lipids- Urinalysis: weekly
LIVER BIOPSIES: after 2 weeks following a 24-hr fast (onlyjuveniles)
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC ANDMICROSCOPIC):
all major organs
STATISTCAL METHODS: student's t test(2) valid with restrictions
Critical study for SIDS endpoint(89)
other: several laboratory animals
no datano dataoral feed
3 days to 3 weeks (see freetext Remarks)
daily
no datasee freetext Remarks
other: see freetext Remarks
1980
no datano data
In a combined species study, species, strain and sex
differences were found. Haemorrhagic deaths were noted inmale rats of strains Sprague-Dawley, Wistar, Donryu andFischer and in female Fischer rats (1.2% BHT in dietcorresponding to between 638-1120 (m) and 854-1000 (f) mg/kg
bw day depending on strain; 3 weeks).
Six different mice strains (only males) were negative inthis respect (847-1925 mg/kg bw day, 1 week), with the
exception of haemorrhagic deaths occurring in ddY strain.
No such effects were seen in dogs (173, 440 or 760 mg/kg bw
day given in diet, 2 weeks), hamsters (380 or 760 mg/kg bw
day i.p., 3 days), guinea pigs (190 or 380 mg/kg bw dayi.p., 3 days), rabbits (177, 242 or 390 mg/kg bw day in
diet, 2 weeks) and quails (1% in diet = ca. 1056 mg/kg bw
day, 17 days). Mice and guinea pigs also showedhaemorrhages, but those did not lead to death.
Haemorrhages were found in the pleural space, musculature,

UNEP PUBLICATIONS

101

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
genitals, nose and intracranial.
Hypoprothrombinaemia without clinical symptoms was in rats,
less markedly in mice, hamsters and quails, but not in
rabbits and dogs. Significant decrease of prothrombin index
in all rats strains (18-92 % of control) and in all mousestrains except ICR mice (79-96 % of control).

Reliability
Flag
19.11.2001
5.5

:
:

Low number of animals; doses reportedly not exact(2) valid with restrictions
Critical study for SIDS endpoint(90)

GENETIC TOXICITY ‘IN VITRO‘

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Ames test
Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538
100-10000 ug/plate

Result:NUMBER OF REVERTANTS PER PLATE in negative control and at
100, 1000, 10000 ug/plate (means of triplicate plates;
positive = 3 times solvent control):- Without metabolic activation:
TA98: 45, 47, 34, 38
TA100: 114, 130, 152, 110
TA1535: 27, 26, 20, 26
TA1537: 5, 7, 7, 5
TA1538: 25, 23, 29, 33

with and without
negative
other: in accordance with Ames et al., Mutat. Res. 31, 347-364 (1975)
1975
no dataother TS: purity ca. 95%, no data about impurities- With metabolic activation:
TA98: 45, 48, 66, 59
TA100: 133, 130, 176, 174
TA1535: 21, 26, 23, 28
TA1537: 4, 6, 6, 10
TA1538: 32, 30, 24, 26
No data about cytotoxicity (but highest concentration 10
mg/plate).
All positive controls valid (with expected responses).

Test condition:

Reliability:

102

EVALUATION OF RESULTS- With metabolic activation: negative- Without metabolic activation: negative
SYSTEM OF TESTING- Type: preincubation method described by Sugimura andNagao, Chemical Mutagens 6, 41-60 (1980)- Metabolic activation system: S-9 mix prepared from liversof Sprague-Dawley rats induced by Aroclor
SOLVENT: DMSOCONTROLS: negative (solvent with and without S-9) andpositive controls (without S-9: sodium azide,
9-aminoacridine, 2-nitrofluorene; with S-9: 2-anthramine)(2) valid with restrictions

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Flag
17.08.2001:

Type
System of testing::

Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

Remark:Result:

Test condition:

Reliability
Flag
08.09.2001

:
:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

HGPRT assay
adult rat liver (ARL) cells, line 18, at passage 25
50, 60, 70, 80, 90 ug/ml
no dataResult:TG resistant mutants per 10E6 colony-forming cells (means)in untreated control, solvent control, positive control andat 50, 60, 70, 80, 90 ug/ml BHT:
53, 67, 334, 45, 66, 77, 51, 67.

Critical study for SIDS endpoint(91)
Bacterial gene mutation assay
S. typhimurium TA102 and TA2638; E. coli WP2/pKM101 and WP2
uvrA/pKM101
0, 333, 525, 1250, 2500, 5000 ug/plate
no cytotoxicity
withnegative
1998
no dataother TS: purity > 95%
A large collaborative study has been performed using thefour bacterial strains in order to compare the specificspectrum of response to chemicals and to evaluate theusefulness of each strain.
Number of revertants/plate not significantly different fromcontrol in any strain.
Plate incorporation method; solvent DMSO; 2 replicates;
positive control; statistical significance analysed using alinear regression test;(2) valid with restrictions
Critical study for SIDS endpoint(92)

negative
other: Tong et al., Mutat. Res. 130, 53-61 (1984)
1984
no dataother TS: purity: ca. 95 %, no data about impurities

Positive control valid.
Test condition:

EVALUATION OF RESULTS: no mutagenicity
METABOLIC ACTIVATION SYSTEM: not applicable (rat liver
cells)SOLVENT: DMSOCONTROLS: negative (without and with solvent) and positive
controls (benzo(a)pyrene)
MEDIA:- WMES medium containing 10% calf serum (no further data)
PERFORMANCE OF TEST:
Log phase cultures exposed for 3 d to the TS, then washed;cultures maintained for 21-23 d for mutant expression,
before they were seeded for selection for HGPRT-deficientmutants; after 24 h WMES containing 20 ug/ml 6-thioguanine(TG; selective medium) was added and cultures re-fed every 4

UNEP PUBLICATIONS

103

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
08.09.2001

:
:

Type
System of testing
Test concentration
Cycotoxic concentr.

:
:
:
:

Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:

Result:days; after 14 d TG-resistant colonies were fixed;
determination of colony-forming efficiency in non-selective
medium, cells fixed after 7-9 d.
(2) valid with restrictions
Critical study for SIDS endpoint(91)
other: chromosome aberration assay
Chinese hamster ovary cells (CHO-W-B1 cells)
1.6, 5, 16 ug/ml
In preliminary growth inhibition tests the high dose reduced growth by 50%(cell survival determined 24 h after treatment)with and without
negative1985
no dataother TS: BHT, no further data
Percent cells with aberrations:- Without metabolic activation:no increased incidence of aberrations
positive control valid- With metabolic activation:
total simple complex aberrations
negative control 3
1
2
low dose550
mid dose871high dose
10
73
positive control 28
16
12

Test condition:

EVALUATION OF RESULTS- Without metabolic activation: not clastogenic- With metabolic activation: slight increase and positive
trend but overall evaluation by the authors: negative
METABOLIC ACTIVATION SYSTEM: S-9 mix prepared from adultmale rat liver induced by Aroclor 1254
SOLVENT: not specified
CONTROLS: negative (solvent) and positive controls(cyclophosphamide with S-9; mitomycin C without S-9)
MEDIA:- Culture medium: McCoys 5a medium supplemented with
L-glutamine, antibiotics, and 10% fetal calf serum

Reliability
Flag
08.09.2001

:
:

Type

:

104

PERFORMANCE OF TEST:-without S-9 TS left in culture until colcemid addition(incubation time 8-12 h, not clearly specified), withmetabolic activation TS added along with S-9 only for 2h at
the beginning of the test period; cells harvested in theirfirst mitosis (determined in SCE studies); 100 metaphases
per group scored; for data analysis aberrations grouped into
categories of simple (breaks and terminal deletions),
complex (exchange and rearrangements) and total; data
evaluation including trend test(2) valid with restrictions
Critical study for SIDS endpoint(93)
other: chromosome aberration assay

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

CHO cells
0.1; 0.25 and 0.5 ug/ml
mitotic index 4.36, 3.83, 3.76 versus 5.60 in DMSO control
without
positive
other: see remark field1995
no dataother TS: BHT from Sigma (no further information)

Remark:Result:Reliability
Flag
08.09.2001::

METHOD:CHO cells were cultured for 15-16 h in the presenceof the different doses of BHT. Two hours before cell
harvesting, cultures were added with colchicine (0.1 ug/ml
final concentration). Air dried slides were preparedfollowing routine protocols. Each treatment was repeated 5
times and a total of 500 metaphases per treatment (100 per
repetition) was scored in coded slides. Statistical analysis
was performed using X2 test. Untreated cultures and DMSO
treated cultures (0.1 ml DMSO per 10 ml culture medium) wereused as controls.
Treatment with the three doses induced a dose dependentsignificant increase of chromatid and isochromatid breaks(5.4, 7.6, 9.2 versus 1.2 breaks per 100 cells; significant
at all concentrations) with a corresponding increase ofabnormal metaphases (6.6, 8.4, 9.4 versus 1.2% in DMSO
control).
(2) valid with restrictions
Critical study for SIDS endpointType
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Sister chromatid exchange assay
CHO cells
0.1, 0.25 and 0.5 ug/ml

Remark:Reliability:

METHOD: For SCE analysis, culture medium was added with 10
ug/ml of 5'-bromo-2'-deoxyuridine (BrdU) and the cells wereincubated in complete darkness. CHO cells were incubated for
30 h. Two hours before fixation, cells were treated withcolchicine (0.1 ug/ml final concentration). For eachtreatment 5 repetitions were made. Air dried slides wereprepared following routine protocols and differential
staining of sister chromatids were obtained according toWolff and Perry (1974). Cytogenetic analysis was performedon coded slides. Statistical analysis was performed using
multifactorial ANOVA. Untreated cultures and DMSO treatedcultures (0.1 ml DMSO per 10 ml culture medium) were used ascontrols.
CYTOTOXICITY: >= 0.25 ug/ml; only a few metaphases could beanalyzed in cells treated with 0.25 ug/ml (23 in relation to
180 of untreated and vehicle controls) and no cells at
second mitosis after 0.5 ug/ml.
(2) valid with restrictions(94)

without
negative
other: see remark field1995
no dataother TS: BHT from Sigma (no further information)

UNEP PUBLICATIONS

105

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Flag
17.08.2001:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Sister chromatid exchange assay
human lymphocytes (from umbilical cord)
0.1, 0.25 and 0.5 ug/ml

Remark:Result:Reliability
Flag
17.08.2001::

METHOD: For SCE analysis, culture medium was added with 10
ug/ml of 5'-bromo-2'-deoxyuridine (BrdU) and the cells wereincubated in complete darkness. Human lymphocytes wereincubated with TS for 24 or 72 h. Two hours before fixation,
cells were treated with colchicine (0.1 ug/ml final
concentration). For each treatment 5 repetitions were made.
Air dried slides were prepared following routine protocols
and differential staining of sister chromatids were obtained
according to Wolff and Perry (1974). Cytogenetic analysis
was performed on coded slides. Statistical analysis was
performed using multifactorial ANOVA. Untreated cultures andDMSO treated cultures (0.1 ml DMSO per 10 ml culture medium)
were used as controls.
CYTOTOXICITY: at 0.5 ug/ml a decrease of cells in second
division with increasing concentration, 35 (24 h) or 26(72h) cells scored in relation to 155 and 165 of untreatedand vehicle controls. 137 (72h) 100 (24h) cells scored at
the low TS concentration and 117 (72h) and 105 (24h) cells
at the mid concentration.
No increase in SCE frequencies detected at any exposure
design.
(2) valid with restrictions
Critical study for SIDS endpointType
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Unscheduled DNA synthesis
rat primary hepatocytes
0.01, 0.1, 0.5, 1.0, 5.0, 10.0 ug/ml
tested up to toxic concentrations

RemarkResult::

Test condition:

Cells with intrinsic metabolic activity.
Valid positive control. No increase in grains/nucleus(triplicate coverslips per concentration) at any
concentration tested compared with the negative control.
Authors evaluation:
BHT did not induce DNA repair in rat hepatocytes exposed up
to toxic concentrations.
Hepatocytes isolated from male F344 rats; cells
simultaneously exposed to the TS and 3H-thymidine for 18 h
and than processed for autoradiography; net nuclear grain

106

Critical study for SIDS endpoint(94)

without
negative
other: see remark field1995
no dataother TS: BHT from Sigma (no further information)(94)

negative
other: Williams et al., Mutat. Res. 97, 359-370 (1982)
1982
no dataother TS: purity: ca. 95 %, no data about impurities

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
counts determined; duplicate experiments; solvent (DMSO) andpositive (2-aminofluorene) control.
(2) valid with restrictions
Critical study for SIDS endpoint

Reliability
Flag
08.09.2001

:
:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: Umu-test
S. typhimurium TA 1535/pSK 1002
0.4, 0.8, 1.5, 3 and 6 mM

Remark:Reliability
Flag
08.09.2001::

METHOD: The umu test was performed by Reifferscheid et al.,Mutat. Res. 253, 215-222 (1991). Salmonella from stock weregrown in nutrient broth for the overnight culture.
Logarithmically growing tester bacteria were exposed tovarying concentrations of the test material. Allconcentrations were tested in triplicate; with each set ofexperiments usually repeated three times. After 2 h of
exposure, the bacterial suspension was diluted 10-fold,followed by a subsequent additional incubation period of 2h. Thereafter, bacterial growth was measured as turbidity(E600) with a microplate reader. The DNA damage induced
expression of umuC was quantified via the determination of
ß-galactosidase activity at 420 nm using ONPG o-nitrophenylß-D-galactopyranoside; Sigma) as a substrate. In all
experiments, the standard genotoxin 4-NQO (4-nitroquinoline
N-oxide) was used as positive control. BHT was dissolved in
DMSO at a stock concentration of 2M. This stock solution wasserially diluted in 1:2 steps and transferred onto themicroplate with the tester organisms using a laboratory
workstation.
CYTOTOXICITY: no(2) valid with restrictions
Critical study for SIDS endpointType
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: review of the mutagenicity/genotoxicity data up to 1991

Result:A host of studies examining the potential of BHT to cause
point mutations have been published. They include in vitro
studies on various bacterial species and strains and on
various types of mammalian cell lines. Together thesestudies convincingly show the absence of a potential for BHT
to cause point mutations: All 19 Ames tests reviewed were
negative as were 3 bacterial gene mutation tests with E.

(91)

without
negative
other: see remark field
1996
no dataother TS: purity: > 98%(95)

UNEP PUBLICATIONS

107

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
coli strains. A mouse lymphoma assay was positive, but onlyat cytotoxic concentrations. A HGPRT test with adult rat
liver epithelial cells was negative. Another HGPRT test with
Chinese hamster V79 cells was positive at slightly cytotoxic
concentrations.
A great number of studies on many cell types have also been
carried out to examine the potential of BHT to cause
chromosome aberrations. In vitro studies have been publishedusing plant cells (4 negative, 1 positive test) and theWI-38 (1 positive, but test system not validated), CHL (2negative), CHO (1 negative, 1 positive, but insufficient
information on cytotoxicity) and V79 mammalian cell lines (1negative). Nearly all studies, especially those using
validated test systems, indicate that BHT lacks clastogenic
potential.
In vitro studies on bacterial cells (5 negative, 1
positive), yeast (3 negative) and various mammalian cells
including DON, CHO, CHL cells and primary hepatocytes (7negative) demonstrate the absence of interactions with ordamage to DNA.
(4) not assignable
Critical study for SIDS endpoint

Reliability
Flag
17.08.2001

:
:

Type
System of testing::

Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

Remark19.11.2001:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Ames test
Salmonella typhimurium TA 97, TA 100, TA 102, TA 104
1 - 1000 ug/plate

Remark:Test substance
19.11.2001:

Cytotoxic levels were tested. No information on purity ofTS.
food grade 2,6-di-tert-butyl-p-cresol; vehicle: DMSO

Type
System of testing
Test concentration

:
:
:

108

(96)
Ames test
Salmonella typhimurium "common" strains (e.g. TA 98, TA 100, TA 1535,
TA 1537 or TA 1538)
up to 10000 ug/platewith and without
negative
other: Various Modifications
no datano data
No information on purity of TS.
(97) (98) (99) (100) (101)with and without
negative
other: Liquid Preincubation Test
1983
no dataother TS

(102)
Bacillus subtilis recombination assay
H 17/M 45
50 µl/paper disk

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:

without
negative
other: Spot Test
1975
no dataother TS: vehicle: DMSO

Remark19.11.2001:

No information on purity of TS.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Bacillus subtilis recombination assay
HA 101no data specified

Remark19.11.2001:

no further data available. No information on purity of TS.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

DNA damage and repair assay
Escherichia coli P37
no data specified

Remark19.11.2001:

No information on purity of TS and concentration tested.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Mitotic recombination in Saccharomyces cerevisiae
D4
no data specified

Remark19.11.2001:

No information on purity of TS and concentration tested.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
Method

:
:
:
:
:
:
:

(99)

with and without
positive
other: no data
1980
no datano data(103) (104)with and without
negative
other: SOS-chromotest
1985
no datano data(105)

no datanegative
other: Gene Conversion Assay1975
no dataother TS: vehicle: DMSO

(100)
Cytogenetic assay
Chinese hamster lung fibroblasts (Don)
2.2 - 220 ug/ml
without
negative
other: Chromosome Aberration Test, BrdUrd-labelling technique

UNEP PUBLICATIONS

109OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Year
GLP
Test substance

:
:
:

1977
no dataother TS: vehicle: DMSO

Remark19.11.2001:

No information on purity of TS.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Sister chromatid exchange assay
Chinese hamster lung fibroblasts (Don)
2.2 - 220 ug/ml

Remark19.11.2001:

No information on purity of TS

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
Chinese hamster fibroblasts (CHL)
2.5 - 75 ug/ml

Remark19.11.2001:

No information on purity of TS.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Sister chromatid exchange assay
Chinese hamster ovary (CHO) cells
1.6 - 16 ug/ml

Remark:Cytotoxic levels were tested. No information on purity of
TS.

(106)

without
negative
other: SCE Test, BrdUrd-labelling technique
1977
no dataother TS: vehicle: DMSO

(106)

without
negative
other: Chromosome Aberration Test
1977
no dataother TS: vehicle: ethanol(107) (108)with and without
negative
other: SCE Test, BrdUrd-labelling technique1985
no datano data

19.11.2001
Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

110

(93)
:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
Diploid human embryonic lung cells (WI-38) in tissue culture
2.5 - 250 ug/ml
250 µg/ml was the highest concentration without cytotoxicity
without
positive
other: Chromosome Aberration Test
1972
no dataother TS: Test substance supplied by US-FDA

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:increase in cells with chromosomal aberrations (mainly
acentric fragments) in anaphase at 24 h after start ofincubation; not dose related(2) valid with restrictions
Critical study for SIDS endpoint

Reliability
Flag
19.11.2001

:
:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
Chinese hamster ovary (CHO) cells
1, 5 and 10 ug/ml
no datawithout
positive
other: Chromosome Aberration Test
1987
no dataother TS: BHT, no further data

Remark:Result:

The incidence of polyploid cells - compared to control - wasincreased but not in a dose-dependent manner;incubation time 48 hours, 100 metaphases per group scored.
TS dissolved in purified absolute ethanol but no data were
given about the ethanol concentration in medium.
The solvent increased the percentage of abnormal cells from
4-6% to 8-12%. No information on cytotoxicity. Contradictory
documentation on statistical evaluation. No information on
purity of TS.
Percent of affected cells(109) (110)untreated ethanol 1 ug/ml BHT
polyploids
6
5
14
aberrations
plus polyploids

6

11

35

5 ug/ml BHT
polyploids

3

3

23

aberrations
plus polyploids

4

5

36

10 ug/ml BHT
polyploids

3

8

12

aberrations
plus polyploids

5

12

20

19.11.2001
Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
Method
Year
GLP

(111)
:
:
:
:
:
:
:
:
:

HGPRT assay
V79 Chinese hamster lung fibroblasts
1, 5 and 10 µg/ml
with
positive
other: no data
1984
no data

UNEP PUBLICATIONS

111

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test substance:no data

Remark:Not selected as key study because positive results wereobtained only at cytotoxic concentrations.

08.09.2001

(112)

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Mouse lymphoma assay
L5178Y2 - 28 ug/ml (with S9-mix); 1.25 - 40 ug/ml (without S9-mix)
see freetext
with and without

Remark:Result:Not selected as key study because no differentiation incolony size and contradictory results in three independent
trials concerning effects; concentrations without effect on
relative total growth did not induce an increase in mutation
frequency (with and without S9-mix).
Cytotoxicity
with S9-mix:
lethal at 14-28 ug/ml (3 trials); RTG < 30% at 10-20 ug/ml(3 trials)
without S9-mix:
lethal at >20 ug/ml (3 trials); relative total growth (RTG)< 30% at 18 ug/ml (1 trial, relative to control).

other: Thymidine Kinase Test
1979
no dataother TS: BHT, no further data

Result with S9 mix: positive
lowest observed effect concentration in one trial 6 ug/ml(RTG ca. 55 % compared to the negative control); in theother 2 trials positive results at RTG < 30% (at 14 and 20
ug/ml).
Result without S9 mix: ambiguous
1 of 3 trials was positive at 18 ug/ml (RTG <30%).
Solvent DMSO, 3 trials, concurrent negative and positive
control(2) valid with restrictions
Critical study for SIDS endpoint

Test condition:

Reliability
Flag
19.11.2001

:
:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: Cell transformation assay
BALB/3T3 clone A31-1-1 cells
10, 20, 25 and 30 ug/ml

Remark:2,6-di-tert-butyl-p-cresol did not act as tumour inductor(72-hour treatment), neither applied alone nor withpromoting treatment with 300 ng TPA (12-O-tetradecanoylphorbol-13-acetate)/ml for 2 weeks after "initiation". No
information on purity of TS.

112

(113)

without
negative
other: 2-Stage Transformation Test (Focus Assay)
1989
no dataother TS: vehicle: DMSOUNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

19.11.2001

(114)

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Escherichia coli reverse mutation assay
WP2
8 - 1000 ug/ml

Remark19.11.2001:

No information on purity of TS

Type
System of testing::

Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:

Remark19.11.2001:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Escherichia coli reverse mutation assay
WP2
no data

Remark19.11.2001:

No information on purity of TS and concentration tested.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: DNA synthesis inhibition test
HeLa S3 cells
0.4, 0.8, 1.5, 3 and 6 mM

Remark:METHOD: In the DIT a culture of logarithmically growing HeLa
S3 cells was transferred into a single cell suspension by
gently detaching the cells with EDTA (250 mg/1 PBS). Thenthe cells were seeded into 96-well microplates at a densitiywithout
negative
other: Preincubation Test
1978
no dataother TS: vehicle: DMSO

(115)
Cytogenetic assay
Barley (Hordeum vulgare) caryopsis and growing root meristematic tips ofonion (Allium cepa)
220.3, 440.6, 881.2 and 1322.0 ug/ml
without
negative
other: Chromosome Aberration Test
1979
no datano data
No information on purity of TS.
(116)

with and without
negative
other: no data
1984
no datano data(117)

without
other: see remark field
1996
no dataother TS: purity: > 98%

UNEP PUBLICATIONS

113

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
of 2 x 10 4 cells/well. The next day, the monolayers of the
HeLa cells were exposed for 90 min to the materials to betested. All concentrations were tested in triplicate; witheach set of experiments usually repeated three times.
Thereafter, the cells were washed by two rinses with fresh,pre-warmed medium and allowed to recover for 2 h. This wasfollowed by addition of BrdU in a final concentration of 20
uM for 60 min. Subsequently, the cells were fixed withethanol/acetic acid/water (90:5:5) for 30 min at room
temperature. The alcohol was poured off and 4 N HCl wasadded to the fixed cells for 10 min to denature the DNA.
Excess acid was washed away by rinsing the microplate twicewith tap water. Then a 1:1500 dilution of a monoclonalanti-BrdU antibody was added to the cells for 30 min. Afterwashing the cells three times with tap water, a 1:500
dilution of perocidase-conjugated F(ab)2-sheep-anti-mouse
IgG antibody was added for another 30 min. The cells were
washed three times with tap water, and a freshly prepared
perocidase sustrate solution was added. The color
development was stopped with a stop solution (H2SO4). The
extinction of the wells was measured at 495 nm using an
ELISA reader. Cell counts were determined by sulforhodamine
B (SRB) adsorption to total cell protein, followed byelution of the dye with Tris buffer and colorimetricmeasurement at 564 nm. In all experiments, the standard
genotoxin 4-NQO was used as positive control. BHT was
dissolved in DMSO at a stock concentration of 2M. This stock
solution was serially diluted in 1:2 steps and transferred
onto the microplate with the tester organisms using alaboratory workstation.
CYTOTOXICITY: >= 1.5 mM; cell count decreased to 32 , 23 and
30% in relation to the vehicle control
limited positive because higher degree of cytotoxicity(cell count < 40%) were observed at concentrations >= 1.5 mM(2) valid with restrictions

Result:Reliability
24.07.2001:

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

other: Anaphase-telophase test
CHO cells
0.1, 0.25 and 0.5 ug/ml
0.25 ug/ml
without

Remark:Result:

Test condition:

Test system not validated. Effects at cytotoxic
concentration only.
CYTOTOXICITY: mitotic index 2.53, 0.9, 0.82 versus 3.17 in
DMSO control.
No significant increase in chromosomal aberrations. At thehighest concentration (compare with cytotoxicity) an
increase (p<0.025) of multipolar mitosis was seen.
METHOD: CHO cells were cultured as monolayer in 24 x 36 mm
cover glasses attached with a small drop of siliconizedgrease to the bottom of 90-mm Petri dishes. Three coverglasses were placed in each Petri dish. Each cover glass wasseeded with 1.5 ml of culture medium containing about 50,000
cells. After 1 h, 8.5 ml of culture medium was added to each

114

(95)

other: see remark field1995
no dataother TS: BHT from Sigma (no further information)

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
Petri dish. Cultures were incubated at 37°C in a humidified
atmosphere of 5% CO2. The set of cultures for eachexperiment was treated simultaneously for 8 h before
fixation to avoid the detachment of cells from cover slides.
Each treatment was repeated 5 times. Cell harvesting was
acomplished by adding an equal volume of fixative(methanol-acetic 3:1) to the culture medium. After 10 min,two changes of fixative were made. Cover glasses were
stained with Carbol fuchsin (Carr and Walker, 1961) andatteched with DPX mounting medium to coded slides.
Statistical comparisons were made by means of the Sokal andRohlf G method (Sokal, 1979). Regression analyzes were
performed to evaluate the mitotic index variations.
Untreated cultures and DMSO terated cultures (0.1 ml DMSO
per 10 ml culture medium) were used as controls.

19.11.2001

(94)

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
CHO cells
1 ug/mlno datawithout
positive

Remark:Result:

Test condition
19.11.2001:

No information on purity of TS; only a single concentration
tested.
Metaphases with at least one chromosomal aberration:
2% (control) versus 4.2%; slight but significant increase.
Solvent DMSO; 500 metaphases per treatment scored.

Type
System of testing
Test concentration
Cycotoxic concentr.
Metabolic activation
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:

Ames test
Salmonella typhimurium TA98, TA100, TA1537
0.05-500 µg/plate
no datawith and without
negative
other: Ames-test
1979
no dataother TS: BHT 99% pure (GC and NMR analysis)

Result:EVALUATION OF RESULTS- With metabolic activation: negative- Without metabolic activation: negative

no dataother TS: BHT (Sigma), no further data

(118)

30.10.2001
5.6

(119)

GENETIC TOXICITY ‘IN VIVO‘

TypeSpecies
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:
:

other: specific locus test
mouse
male
other: (C3Hx101)F1
oral feed

UNEP PUBLICATIONS

115

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:

0.75% BHT in the diet (corresponding to ca. 1000 mg/kg bw day)negative

Result:

Test condition:

Reliability
Flag
26.03.2003::

The mean interval between start of administration of
BHT-containing diet to parents and conception of offspringwas 368 days.
Matings with untreated females resulted in 3928 litters,
mean litter size 5.2(within the normal range of the lab); only one mutation
among 20402 offspring alive at weaning (background incidence
28/531500).
Author's conclusions: BHT is not likely to induce point
mutations in germ cells; based on worst case assumptions,they estimated that for current human exposure Levels
mutation induction by BHT of 8% or more over backgroundmutation frequency is excluded with 95% confidence.
206 exposed male mice caged with T-stock females during
their reproductive life (in most cases more than 2 years);
age at study intitiation 8-10 weeks; exposure started 30
days prior to mating; mating period 119 weeks; females
replaced after 1 year or earlier if infertile; offsprings
scored at weaning
for specific locus mutations by means of 7 genetic markers.
(2) valid with restrictions
Critical study for SIDS endpoint

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Micronucleus assay
mouse
femaleother: hybrid mouse, genotype (C57BL/6 x C3H/He)i.p.
5 days (daily injections)0, 125, 250, 500, 1000 mg/kg bw day
negative

Result:

Test condition:

The frequency of micronuclei was not increased at any doselevel.
Highest dose ca. 1/2 of the LD50; 8 mice per group (4 males,
4 females); 11-14 weeks old; vehicle control (DMSO); micekilled on the 5th day 4 h after the last injection; ca. 1000
bone marrow reticulocytes per group scored (333 per animal;
hence, 3 animals per dose were used).

1976
no dataother TS: BHT, no further data

(120)

1979
no dataother TS: BHT 99% pure (GC and NMR analysis)

Positive controls: not specifically noted, but 19 of the 61
substances tested in this study were positive.

Reliability

116:

Evaluation:
result considered positive when the treated group exceeded
the control group by 1% (10/1000) which was ca. double ofthe control.
(2) valid with restrictions

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Flag
26.03.2003:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Micronucleus assay
mouse
male/female
other: (CBAxC57BL/6J)F1
i.p.
once
75 mg/kg bw
negative
other: Schmid (1973) Agens & Actions 3: 77

Result:

Test condition:

Reliability
Flag
17.08.2001::

Incidence of micronuclei in polychromatic erythrocytes was
not statistically different from that in the control at alltime points:
Control: 1.7+/-0.4 (24h); 1.7+/-0.5 (48h); 1.7+/-0.6 (72h);
1.4+/-0.7 (96h)Treated group: 2.6+/-0.3 (24h); 1.3+/-0.8 (48h); 1.6+/-0.3(72h); 1.5+/-0.3 (96h)
5 mice (60-75 days old; body weight 27-30 g) per treated andcontrol group.
Bone marrow sampled 24, 48, 72, and 96h after application;
1000 polychromatic erythrocytes per animal scored.
Rate of spontaneous micronuclei stable in the hybrid strain
used: less than 2 per 1000 cells.
Statistical analysis: Student's t test(2) valid with restrictions
Critical study for SIDS endpoint

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
ratmaleother: Wistar and Sprague-Dawleyoral feed
9 months
0 or 1.5 % in diet (ca. 750 mg/kg bw day)negative

Result:Number of cells (250 cells examined per group) withaberrations:

Critical study for SIDS endpoint(119)

no dataother TS: synthesized by Institute of Chemical Physics, Academy of
Sciences of the USSR (purity not specified)(121)

no dataother TS: BHT, product of Tokyo Kasei Kogyo Co., Ltd., no further data

Chromatid break Chromatid Gap Other
Wistar rats
control
1
3
0
BHT
0
2
0
SD rats
control
0
4
0
BHT
0
2
0
Evaluation:

UNEP PUBLICATIONS

117

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
no adverse effects on chromosomes.
5 rats/strain/group; rats were 5 weeks old at the start of
the treatment period; 2 h before sacrifice rats treated withcolchicine; bone marrow cells prepared; 50 metaphases per
animal checked for chromosomal aberrations.
Positive control: not reported(2) valid with restrictions
Limitations: 1 dose, positive control not reported
Critical study for SIDS endpoint

Test condition:

Reliability:

Flag
26.03.2003:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Cytogenetic assay
mouse
male
ICR
oral feed
9 months
0 or 1.5 % in diet
negative

Result:Number of cells (250 cells examined per group) withaberrations:(115)

no dataother TS: BHT, product of Tokyo Kasei Kogyo Co., LTD, no further data

control
BHT

Test condition:

Reliability:

Flag
26.03.2003:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

118

Chromatid break Chromatid Gap Other
2
5
0
0
3
0

Evaluation:no adverse effects on chromosomes.
5 mice/group, 5 weeks old at the start of the treatment
period; 1.5 % in diet (report states same intake as rats,
i.e. 750 mg/kg bw day; probably typographic error; based onstandard diet conversion factor 1,5% correspond to ca. 2000
mg/kg bw day)2 h before sacrifice rats treated withcolchicine; bone marrow cells prepared; 50 metaphases per
animal checked for chromosomal aberrations.
Positive control: not reported(2) valid with restrictions
Limitations: 1 dose, positive control not reported; generalreporting deficiences
Critical study for SIDS endpoint(115)
other: Chromosome Aberration Testrat
maleother: random bred albino
gavagefive administrations, 24 h apart30, 250 or 500 mg/kg bw day
negative
other: Metaphase analysis of bone marrow cells
1972
no dataother TS: Test substance supplied by US-FDA

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Metaphases examined once (6 h after last administration); 5
animals per dose group; 3 animals in negative control group.
(2) valid with restrictions
Critical study for SIDS endpoint

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: Chromosome Aberration Testrat
maleother: Random bred albino
gavagesingle administration
30, 900 and 1400 mg/kg bw
negative
other: Metaphase analysis of bone marrow cells
1972
no dataother TS: Test substance supplied by US-FDA

Remark:Reliability
Flag
26.03.2003::

Metaphases examined 6, 24 and 48 hours after single
administration; 15 animals per dose group, 5 animals
sacrificed at each time point; 3 animals in negative control and 9 animals in
positive control group (0.5 mg/kg bw TEM i.p.)(2) valid with restrictions
Critical study for SIDS endpoint(109) (110)Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: Chromosome Aberration Test
rat
no datano dataother: no data
no datano datanegative

Reliability:(4) not assignable
No information on test procedure(109) (110)1980
no datano data

29.10.2001

(104)

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Dominant lethal assay
ratmaleSprague-Dawleyoral feed
10 weeks
0.04, 0.13 and 0.4 % (w/w) in dietResult:Toxicity:
significantly decreased body weight gain in the high dose

(50, 166 and 500 mg/kg bw day)no dataother TS: BHT, no further data

UNEP PUBLICATIONS

119OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
group.
Dominant lethal effects:
significant increases were found in dead implants
per pregnant females (at 0.13 and 0.4 % in diet mated in thefirst week), and in the number of pregnant females with morethan 1 - 2 dead implants (at 0.13 % in diet, mated in thefirst week). The number of live implants per pregnant female
was slightly decreased (significant at 0.13 and 0.4 % in
diet, respectively mated in the second week).
In this study the results were considered positive withregard to the rate of dead implants.
But recent calculations of the total dominant lethal effectare based on comparison of the live implants per female in
the test groups to the live implants per female in thecontrol group. According to this kind of evaluation the
results of the present study have to be considered negative.
MTD determined in a 5-day gavage study; TS incorporated into
the diet with the aid of corn oil (final concentration ofcorn oil in the diet 3%); TS diet levels without adjustment
to changes in body weight or food consumption; negative
vehicle control and positive control (triethylenemelamine);
diet prepared twice weekly; after 10 weeks treatment each of
20 male rats per group mated with 2 virgin females per week
for 2 successive weeks; females sacificed 14 d after midweek
of mating and number of dead and live implants counted.
(2) valid with restrictions
Critical study for SIDS endpoint(122) (123) (124) (125) (126)

Test condition:

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Dominant lethal assay
mouse
male
ICR
oral feed
9 months
1.5 % in diet (ca. 750 mg/kg bw day)Remark:Result:control: 9 males, mated with 18 females;
treated group: 10 males, mated with 20 untreated females. No information
on purity of TS.
Compared with the untreated control group, no statisticallysignificant difference was obtained in copulation rate,
pregnancy rate, mortality of eggs, embryos and foetuses,
number of living foetuses, and induction rate of dominantlethality.
(127)

26.03.2003
Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses

120

:
:
:
:
:
:
:

other: Dominant Lethal Assayno datano data

Dominant lethal assay
mouse
male
other: ICR/Ha Swissi.p.
single injection
250 - 2000 mg/kg bw

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result
MethodYear
GLP
Test substance

:
:
:
:
:

other: Dominant Lethal Test
1972
no dataother TS: vehicle: tricaprylin or distilled water

RemarkResult19.11.2001::

No information on purity of TS
negative

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Dominant lethal assay
mouse
male
other: (101 x C3H)
oral feed
8 weeks
1 % (w/w) in diet (ca. 1500 mg/kg bw day)Remark:Result26.03.2003:

Males were mated with (SEC x C57BL) and (C3H x C57BL)
females. No information on purity of TS.
negative

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Dominant lethal assay
ratmaleSprague-Dawley
gavage5 administrations 24 hrs apart30, 250 and 500 mg/kg bw day
negative
other: Dominant Lethal Test
1972
no dataother TS: TS supplied by US-FDA

Remark:Result:Reliability
Flag
26.03.2003::

Negative control and positive control (0.2 mg/kg bw TEM i.p. once); 10
males treated per dose group; after the fifth dose weekly mating with 2
untreated virgin females each for 7 weeks; 1/4 of the pregnant females ineach dose group were sacrificed on each of the 4 days starting on the 15th
day after the 1st day of breeding. This schedule allowed for sacrifice offemales between days 11 and 18 of gestation.
Complete autopsy of each female was done.
No consistent responses which could be attributed totreatment.
(2) valid with restrictions
Critical study for SIDS endpoint(109) (110)Type
Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:
:

(128)

other: Dominant Lethal Test
1986
no dataother TS: vehicle: corn oil(125)

Dominant lethal assay
ratmaleSprague-Dawley
gavagesingle administration

UNEP PUBLICATIONS

121

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:

30, 900 and 1400 mg/kg bw
negative
other: Dominant Lethal Test
1972
no dataother TS: TS supplied by US-FDA

Remark:ResultReliability
Flag
26.03.2003:::

Negative control and positive control (0.2 mg/kg bw TEM i.p. once); 10
males treated per dose group; within 2 to 3 hrs after dosing each male wasmated with 2 untreated virgin females for 7 days; females replaced weekly
for total mating period of 8 weeks. 1/4 of the pregnant females in eachdose group were sacrificed on each of the 4 days starting on the 15th day
after the 1st day of breeding. This schedule allowed for sacrifice of females
between days 11 and 18 of gestation. Complete autopsy of each female
was done.
negative(2) valid with restrictions
Critical study for SIDS endpoint(109) (110)Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Mammalian germ cell cytogenetic assay
mouse
male
other: hybrid mouse, genotype (C57BL/6 x C3H/He)i.p.
5 days (daily injections)
125, 250, 500 or 1000 mg/kg bw day

RemarkResult::

Test condition:

No information on purity of TS
Low non-dose related incidence up to 3.5% (controls up to1%) of abnormal spermatozoa (maximum effect at 250 mg/kg bw day).
4 animals per group
Sacrifice on day 35 after last injection
Examination of 333 sperms from each animal.
(119)

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

Heritable translocation assay
mouse
male
other: (101xC3H)F1
oral feed
8 weeks
0 or 1% BHT in the diet
negative
other: Generoso et al., Mutat. Res. 73, 133-142 (1980)1985
no dataother TS: BHT, no further data

Result:Toxicity:no adverse effects observed with this dose; in preliminaryexp. 1.5% revealed reduced body weight gain, at 3% all micedied.

26.03.2003

other: Sperm Abnormality Test
1979
no dataother TS: vehicle: DMSO

Induction of heritable translocations in males:

122

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test condition:

Reliability
Flag
08.09.2001

:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
Method

:
:
:
:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:ResultReliability
Flag
26.03.2003

:
:
:

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
Method

:
:
:
:
:
:
:
:
:

Year
GLP
Test substance:::

Remark:ResultReliability
Flag
26.03.2003:::

Control: 503/507 (99.4%) normal in fertility testing,treatment group: 473/475 (99.6%) normal in fertility
testing.
Evaluation:
under the condition of the study, BHT is negative forinduction of heritable translocations in male mice.
Before TS treatment male mice mated with 3 (SECxC57BL)F1
females for 1 week; thereafter males treated with 1% TS in
the diet for 8 weeks (n=50 per group); after exposure period
males again mated with the same group of females; males ofprogeny produced before and after TS treatment subjected tofertility procedure; males with reduced fertility subjected
to cytological analysis for the confirmation oftranslocation heterozygosity.
Total of 507 control and 475 exp. male progeny tested.
(2) valid with restrictions
Critical study for SIDS endpoint(125)
other: Host-mediated assay
other: Saccharomyces cerevisiae
other: D3
gavage5 applications
30, 250 and 500 mg/kg bw day of hostnegative
other: according to Gabridge and Legator, Proc. Soc. Exp. Biol. Med. 130,831 (1969)1972
no dataother TS: TS supplied by US-FDA
host: Swiss Webster male mice (10 animals per dose group);
positive control: 350 mg EMS/kg bw i.m.)
negative(2) valid with restrictions
Critical study for SIDS endpoint(109) (110)other: Host-mediated assay
other: Salmonella typhimurium
other: TA 1530, G 46
gavagesingle application
30, 900 and 1400 mg/kg bw of hostnegative
other: according to Gabridge and Legator, Proc. Soc. Exp. Biol. Med. 130,831 (1969)1972
no dataother TS: TS supplied by US-FDA
host: Swiss webster male mice (10 animals per dose group);
positive control: 10 mg DMNA/kg bw i.m.)
negative(2) valid with restrictions
Critical study for SIDS endpoint(109) (110)UNEP PUBLICATIONS

123

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: in vivo-in vitro replicative DNA synthesis test
rat
maleFischer 344
other: gavage or s.c. injection (no further information available)
single dose
450 mg/kg bw and 900 mg/kg bw
positive
other: see remark field1994
no datano data

Remark:Result:Reliability
26.03.2003:

METHOD: the vehicle used was corn oil; the numbers ofanimals treated and the number from which primary hepatocyte
cultures were produced is not mentioned; production ofprimary hepatocyte cultures and assessment of RDS induction
was performed using published procedures (Uno et al.,
Toxicol. Lett. 63, 191-199 and 201-209 (1992));
Judgement criteria for RDS incidence: RDS incidence was
evaluated by our earlier documented judgement criteria. In
the time-course experiment, when the maximum RDS incidence
was 2.0% or above, it was considered to indicate a positive
response. An incidence less than 1.0% was judged to be
negative. an incidence between 1.0 and 2.0% was considered
equivocal, and a dose-response experiment was subsequently
performed. In this second experiment, when the incidence was
1.0% or above at any of the doses, a final judgement of
positive was made, whereas a reponse of less than 1.0% was
rated as negative.
In the time course experiment BHT caused dose-related RDS
induction; RDS incidence (%) after 450 mg/kg bw: 0.3 (24 h), 1.2 (39 h),
0.2 (48 h); RDS incidence (%) after 900 mg/kg bw: 2.5 (24 h), 9.2 (39 h),
0.8 (48 h) the hepatocyte viability did not vary from untreated control value(3) invalid
(129)

Type
Species
Sex
StrainRoute of admin.
Exposure period
Doses
Result
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:

other: liver DNA damage
rat
femaleSprague-Dawley
gavagefirst dose 21 h before killing; second dose 4 h before killing
among others 700 mg/kg bw and 140 mg/kg bw (no further information)

Remark:METHOD: the vehicle used for gavage was 2% gum tragacanth inwater; the numbers of animals treated and the number fromwhich hepatic DNA was otained is not mentioned; the rat
hepatic DNA damage assay (alkaline elution) was performed as
described by Kitchin and Brown, Teratogenesis,
Carcinogenesis and Mutagenesis 9, 61 (1989). The data wasanalyzed by analysis of variance, and where statisticallysignificant differences wer found, they were then evaluatedwith Student´s t-test.

124

other: see remark field1994
no datano data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:Reliability
26.03.2003:

5.7

As the highest dose did not show the DNA-damaging effects
that one lower dose did, no dose response curve or
regression model will fit;
the highest tested dose that did not cause rat liver DNA damage to astatistically significant extent: 140 mg/kg bw;
the lowest tested dose that caused rat liver DNA damage: 700 mg/kg bw.
(3) invalid(130)

CARCINOGENICITY

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group

:
:
:
:
:
:
:
:
:
:

MethodYear
GLP
Test substance

:
:
:
:

Remark:Result:mouse
femaleCD-1
dermal
5 weeks
twice weekly
none
10 mg/application/animalother: concurrent, no initiation or no promotion treatment or vehicle
treatment only
other: Initiation/Promotion Assay1987
no dataother TS: purity: 98 %; vehicle: DMSO
Initiation treatment: 2,6-di-tert-butyl-p-cresol was
topically applied onto the shaved backs of 15 animals.
Promotion treatment: Topical application of 2.5 ug
12-O-tetradecanoyl phorbol-13-acetate/animal onto theinitiation treatment site twice weekly for 47 weeks starting
1 week after the last initiation treatment.
Control groups: No initiation treatment (18 animals), no
promotion treatment (14 animals), vehicle treatment only
(20 animals). Low number of animals per dose group.
2,6-Di-tert-butyl-p-cresol did not show tumour initiation
activity in mouse skin.

19.11.2001(131)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
femaleSprague-Dawleyi.p.
3 weeksdaily
20 weeks
200 mg/kg bw day

Remark:Carcinogen treatment: Single intubation of approx. 5 mg
7,12-dimethylbenz(alpha)anthracene (DMBA)/animal.
Inhibition treatment: 2,6-di-tert-butyl-p-cresol (BC)administration started 2 weeks prior to carcinogen treatment

yes, concurrent vehicle
other: Mammary Tumour Prevention Assay1989
no dataother TS: vehicle: corn oil

UNEP PUBLICATIONS

125OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:Source26.03.2003:

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark:Result:126(25 animals).
Metabolite treatment: In order to investigate the mode ofaction in mammary tumour prevention effect additional
studies were carried out with two of the major oxidative BC
metabolites, 2,6-di-tert-butyl-4-hydroxymethylphenol (BCOH) and 2,6-di-tert-butyl-1,4-benzoquinone (BC-quinone),
following the same treatment regimen as was done with BC.
No information on purity of TS.
The extent of tumour inhibition by BC (= 39 %) was greaterthan that exhibited by BC-quinone (= 25 %) while theadministration of BC-OH did not inhibite mammary
tumourigenesis. Thus, the inhibition of DMBA-induced mammary
tumourigenesis by BC does not appear to be mediated by theoxidative BC metabolites BC-OH or BC-quinone.
Shell
(132)
rat
male/female
Wistaroral feedin utero, during lactation and until 141 - 144 weeks of age
daily
none
in utero: 25, 100 and 500 mg/kg bw of pregnant female/day (parental
females: until the end of lactation);after weaning: 25, 100, 250
mg/kg bw day
yes, concurrent no treatment
other: In utero Cancerogenicity Study
1986
no dataother TS
Among the non-neoplastic lesions in the liver, a doserelated increase in the incidence of bile-duct proliferationand cysts was found in males and of focal cellular enlargement in females.
Average food consumption was unaffected by treatment, butmean body weight was depressed in a dose-related manner atthe end of the lactation period, an effect persisting
throughout the study in both sexes (being most pronounced inmales).
Treated rats obviously survived longer than the controls.
No treatment-related changes were found in haematologicalparameters.
Females treated with the highest dose showed an increase inserum cholesterol and phospholipids, whereas serum triglycerides were reduced in this group in both sexes.
80, 80 and 100 rats/sex, respectively in the 25, 100 and 250
mg/kg bw day dose groups; 100 rats/sex were fed the control diet;the groups were formed from 29, 30 and 44 litters of therespective parental dose groups and from 40 litters of theparental control. The parental males and females werealready dosed during 13 weeks of premating. The F1 250 mg/kg
group derived from parentals exposed to 500 mg/kg bw day (the dose
was lowered because of adverse effects on the kidney in theparental females).
Data concerning reproduction parameters of this study are
depicted in chapter 5.8.
Dose-related increases in hepatocellular carcinomas (1 %, 0 %, 1 %, 8 %UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
at 0, 25, 100, 250 mg/kg bw day) and hepatocellular adenomas (1 %, 1 %,6 %, 18%) for male rats; the increased incidences for hepatocellularadenomas andcarcinomas at 250 mg/kg bw day were significant. Trend analysis also
revealed a dose-effect relationship for both, adenomas and carcinomas.
Dose-related increases in the numbers of hepatocellular adenomas andcarcinomas in treated females were only statistically significant foradenomas.
All hepatocellular tumours were detected when the rats weremore than 2 years old. Tumors were found in many otherorgans of some of the treated rats, but their incidence was
not significantly different from that in controls.
The validity of the significancies is uncertain because of
the increased survival in the treated groups compared to thecontrol group; the authors stated, that the role of
2,6-di-tert-butyl-p-cresol in the development of hepatocellular tumours requires further elucidation.
food-additive grade; purity: > 99.5 %(2) valid with restrictions
Critical study for SIDS endpoint(79)

Test substance
Reliability
Flag
26.03.2003:::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male/female
Fischer 344
oral feed
105 weeks
daily
none
3000 and 6000 ppm (ca. 225 and 450 mg/kg bw day)Remark:Result:Reliability
Flag
26.03.2003::

Body weight gain was reduced in dosed rats; survival was notaffected by test substance treatment;
50 rats/sex and dose group, control: 20 rats/sex.
No significantly higher incidences of tumours were found ineither male or female rats.
(1) valid without restriction
Critical study for SIDS endpoint

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

yes, concurrent no treatment
other: Carcinogenicity Study
1979
no dataother TS: purity: 99.9 %

(133)
rat
male
Wistaroral feed
32 weeksdailynone
1 % (w/w) in diet (ca. 500 mg/kg bw day)other: concurrent, no initiation and/or no promotion treatment
other: Gastric Tumour Promotion Assay1986
no dataother TS: purity: 98 %UNEP PUBLICATIONS

127

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Result:Initiation treatment: 100 mg N-methyl-N'-nitro-N-nitrosoguanidine/ml in the drinking water for an 8-week periodprior to promotion treatment.
Promotion treatment: Subsequent to the initiation treatment,
20 animals were fed 2,6-di-tert-butyl-p-cresol.
Control groups: Initiation treatment only (18 animals), notreatment at all (10 animals), 2,6-di-tert-butyl-p-cresol
administration without previous initiation treatment(10 animals). No information on purity of TS.
Neither the overall tumour incidence nor the specificlocalization of tumours in the glandular stomach weresignificantly changed by the 2,5-di-tert-butyl-p-cresol
treatment.

26.03.2003(134)

Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:

Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:

Remark:Result:rat
male
Wistaroral feed
9 days (with concomitant initiation treatment) or 24 weeks (subsequent toinitiation treatment)
daily
none
1.0 % in diet (ca. 500 mg/kg bw day)other: concurrent, no initiation and/or no promotion treatment
other: Initiation/Promotion Assay1989
no datano data
Initiation treatment: Two injections of azaserine (AZA) at adose level of 30 mg/kg bw each at a 6-day interval.
Promotion treatment: 2,6-di-tert-butyl-p-cresol (BC)administration, starting two days prior to the firstinitiation treatment (concomitant initiation treatment = BC
+ AZA group) or starting one day subsequent to the second
initiation treatment (AZA + BC group), respectively.
Parameters: Glutathione S-transferase placental form (GST-P)
positive foci in the liver, glutathion S-transferase A form
(GST-A) positive foci in the pancreas and in the kidney. No
information on purity of TS.
BC + AZA: No effect on GST-P positive foci/cm2 in the liverand on GST-A positive foci/cm2 in the kidney; significantly
reduced GST-A positive foci/cm2 in the pancreas.
AZA + BC: No effect on GST-A positive foci/m2 in thekidney; significantly reduced GST-P positive foci/m2 in theliver; significantly increased GST-A positive foci/cm2 in
the pancreas (corresponding results with respect to focisize).
The results point to a marked difference in the response ofliver and pancreas, while demonstrating significant second
stage promotion of pancreatic acinar carcinogenesis by
2,6-di-tert-butyl-p-cresol.

26.03.2003
Species
Sex
Strain

128

(135)
:
:
:

rat
male
Wistar

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Route of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:

oral feed3 - 22 weeks
daily
none
0.5 % in diet (ca. 250 mg/kg bw day)Remark:Result:Initiation treatment: Single i.p. injection of 200 mg diethylnitrosamin/kg bw.
Selection treatment (starting 2 weeks after the initiation
treatment): rats were fed a 0.03 % 2-acetylaminofluorene(2-AAF) containing diet for a 2-week period, a single intragastric dose of 2 ml CCl4/kg was administered after 1 weekof 2-AAF feeding.
Promotion treatment: Starting 1 week after termination of
selection treatment. 8 - 10 animals were sacrificed,
respectively after 3, 6, 14, and 22 weeks of administrationof the 2,6-di-tert-butyl-p-cresol diet and livers were
examined.
Parameter: Gamma-glutamyltransferase (GGT) positive foci.
No information on purity of TS.
Histochemical findings: Percentage of liver parenchyma
occupied by, and number per cm2 of GGT positive foci wereincreased slightly after 3 and 6 weeks and strongly after
14 weeks of promotion treatment compared to control.
Gross pathology: Cancer incidence (1/10) was in the range ofcontrol (1/9) including one cholangiocarcinoma and one
hemangiosarcoma at termination after 22 weeks of promotion
treatment.
Histopathology: Mainly eosinophilic foci and nodules, someoval cell proliferation and additional cholangiomas, cystic
changes and telangectasia were observed.

other: concurrent, no promotion treatment
other: Initiation-Selection/Promotion Assay1983
no datano data

26.03.2003(136)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark:rat
maleFischer 344
oral feed
22 weeksdaily
none
0.25, 0.5 or 1.0 % in dietbw day)(ca. 125, 250 or 500 mg/kg

other: concurrent, no initiation or no promotion treatment
other: Bladder Carcinogenesis Promotion Assay1987
no dataother TS: purity: > 99 %Initiation treatment: N-Butyl-N-(4-hydroxybutyl)nitrosamine(BBN) at a concentration level of 0.05 % in the drinking
water for 2 weeks.
On day 22 of the experiment, the lower section of the left
ureter of all rats was ligated.

UNEP PUBLICATIONS

129

OECD SIDS
5. TOXICITY

Result2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

Promotion treatment: 14, 13 or 17 animals received 2,6-ditert-butyl-p-cresol (BC) at low, intermediate or high doselevel, respectively.
Control groups: Initiation treatment only (14 animals);
500 mg BC/kg without previous initiation treatment(17 animals).
Surviving rats were killed at the end of week 24 of thestudy.
Parameter: Urinary bladder papillary or nodulary hyperplasia
counted by light microscopy.
Uncommon study design.
2,6-di-tert-butyl-p-cresol increased dose-dependently theincidence and number of preneoplastic lesions of the urinary
bladder (papillary or nodular hyperplasia) in rats treatedwith BBN. The effect was significant in the high dose groupconcerning both incidence (as percentage) and number ofhyperplasia/10 cm basement membrane.

26.03.2003(137)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344/DuCrj
oral feed
32 weeksdaily
none
0.8 % in diet (ca. 400 mg/kg bw day)Remark:Result:Initiation treatment: N-butyl-N-(4-hydroxybutyl)nitrosamine
was administered at a concentration level of 0.05 % in thedrinking water for 4 weeks.
Promotion treatment: 16 animals received 2,6-di-tert-butylp-cresol after termination of initiation treatment.
Control group: 20 animals.
No informations on purity of TS.
Significantly increased density of papillary or nodulary
hyperplasia in the bladder (3.3 +- 2.6/10 cm basement
membrane in comparison to 1.3 +- 1.6/10 cm basement membranein the control group), whereas the incidence was not
significantly different from control value. There were no
significant differences in both incidences and densities of
bladder papillomas and carcinomas in comparison to control
group.

other: concurrent, no promotion treatment after initiation
other: Bladder Carcinogenesis Promotion Assay1989
no datano data

26.03.2003(138)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

Result

:

130

rat
male
Fischer 344
oral feed
76 weeksdaily
none
100, 300, 1000, 3000 or 6000 ppm in diet
300 mg/kg bw day)

UNEP PUBLICATIONS(ca. 5, 15, 50, 150 orOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Control group
MethodYear
GLP
Test substance

:
:
:
:
:

other: concurrent, no initiation and/or no promotion treatment
other: Liver and Bladder Carcinogenesis Promotion Assay1991
no dataother TS: purity: 95 %Remark:Result:Initiation treatment: 2-Acetylaminofluorene (2-AAF) at aconcentration level of 50 ppm in the diet for 76 weeks.
Promotion treatment: 36 animals/dose group received
2,6-di-tert-butyl-p-cresol (BC) simultaneously with theinitiation treatment.
Control groups: No treatment at all or initiation treatment
only (36 animals/control group), BC administration (6000ppm) without initiation treatment (21 animals).
Parameters: In all groups 3 - 4 randomly selected rats were
killed at weeks 12, 24, 36 and 48 for histochemical evaluation and morphometric analysis of gamma-glutamyltransferase(GGT) positive and iron-storage (IS) deficient foci in theliver. At 76 weeks the experiment was terminated and all
remaining rats were killed. Hepatocellular neoplasms andurinary bladder tumours and lesions were histologicallydiagnosed.
Low number of animals per group.
Simultaneous feeding of BC inhibited the induction ofaltered liver foci by 2-AAF in a dose-related manner (ofboth GGT positive and IS deficient foci with respect tonumber as well as size and percentage of area) and reduced
the incidence of hepatocellular carcinomas and the number ofliver neoplasms per animal.
Feeding of 6000 ppm BC, but not of lower doses, together
with 2-AAF resulted in an increase in the incidence andmultiplicity of bladder neoplasms, and 3000 ppm increased
nodular hyperplasia in the bladder.

26.03.2003(139)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
32 weekdaily
none
1 % in diet (ca. 500 mg/kg bw day)Remark:Result:Initiation treatment: N,N-Dibutylnitrosamine at a concentration level of 0.05 % in the drinking water for 4 weeks.
Promotion treatment: 21 animals received 2,6-di-tert-butylp-cresol after termination of initiation treatment.
Control groups: Initiation treatment only (21 animals),
2,6-di-tert-butyl-p-cresol treatment without previous
initiation treatment (20 animals).
No informations on purity of TS.
2,6-di-tert-butyl-p-cresol significantly increased theincidence of esophageal carcinoma and papilloma but did notother: concurrent, no initiation or no promotion treatment
other: Esophagus and Forestomach Carcinogenesis Promotion Assay1987
no dataother TS: food additive grade

UNEP PUBLICATIONS

131OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
enhance forestomach carcinogenesis. (In addition 2,6-ditert-butyl-p-cresol tended to increase DNA synthesis ofesophageal epithelium but not that of the forestomach as wasinvestigated in rats after a 2-week promotion treatment
period by BrdUrd/immunohistological technique.)26.03.2003(140)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
16 weeksdaily
none
0.7 % in diet (ca. 350 mg/kg bw day)Remark:Result:Initiation treatment: N,N-Dibutylnitrosamine (DBN) at aconcentration level of 0.05 % in the drinking water for 16
weeks.
Promotion treatment: 16 animals received 2,6-di-tert-butylp-cresol (BC) simultaneously to the initiation treatment.
Control groups: Initiation treatment only (DBN, 20 animals),
2,6-di-tert-butyl-p-cresol treatment without previous
initiation treatment (BC, 19 animals).
No informations on purity of TS.
The incidence of hepatocellular carcinomas (16/16) andmetastases in the lung (8/16) was significantly increased
in comparison to the DBN control group (8/20 and 0/20,
respectively). On the other hand, the incidence of hyperplasia in the forestomach (2/16) was decreased in comparison
to the DBN control group (13/20).

other: concurrent, no initiation or no promotion treatment
other: Liver, Forestomach and Bladder Carcinogenesis Promotion Assay1988
no dataother TS: food additive grade

26.03.2003(141)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
femaleSprague-Dawleyoral feed
180 daysdaily
none
5000 ppm in diet (ca. 250 mg/kg bw day)Remark:Carcinogen treatment: Single intragastric instillation of
16 mg 7,12-dimethylbenz(alpha)anthracene (DMBA)/animal.
Inhibition/combination treatment:
30 animals/group received either
2,6-di-tert-butyl-p-cresol (BC) or retinyl acetate (RA) at a dose level of 250
mg/kg bw day with the diet or simultaneous administrations of BC and RA

132

other: concurrent, see remark
other: Mammary Tumour Prevention Assay1986
no datano data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

Result2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

26.03.2003
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark:Result:for a 3-weekperiod starting 2 weeks prior to DMBA application or for a
180-day period starting either 2 weeks prior to or 1 week
after DMBA application.
Control groups: No treatment at all, only BC or only RA
inhibition treatment, simultaneous combination treatment
with both BC and RA without carcinogen treatment(10 animals/group).
No informations on purity of TS.
BC and RA were both effective in the prevention of mammary
cancer induction. Combined administration of BC plus RA was
more effective in mammary cancer chemoprevention than was
BC or RA alone and the interaction of both substances wasadditive. Chronic exposure to BC plus RA induced a highincidence of hepatic fibrosis and bile duct hyperplasia that were notobserved in controls but were seen in low incidence in animals exposed to
BC or RA only.
(142)
rat
femaleSprague-Dawleyoral feed
210 daysdaily
none
300, 1000, 3000 or 6000 ppm in dietmg/kg bw day)other: concurrent, no inhibition treatment
other: Mammary Tumour Prevention Assay1986
no dataother TS: food grade, purity: 99.94 %Carcinogen treatment: Single intragastric administration of
5 or 15 mg 7,12-dimethylbenz(alpha)anthracene (DMBA)/animal.
Inhibition treatment: 2,6-di-tert-butyl-p-cresol (BC)administration started 2 weeks prior to carcinogen treatment(number of animals varied between 39 and 60 for thedifferent concentration level groups).
Control groups: number of animals varied between 21 and 30.
Low number of animals per dose group.
A significant overall inhibitory trend in mammary tumour
incidence was observed. The degree of carcinogenesis
suppression depended on the concentration level of BC in thediet and on the dose of carcinogen administered.

26.03.2003
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear(ca. 15, 50, 150 or 300

(143)
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
11 weeksdaily
44 weeks
1.0 % in diet (ca. 500 mg/kg bw day)other: concurrent, no promotion treatment
other: Multi-Organ Initiation/Promotion Assay
1991

UNEP PUBLICATIONS

133

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

GLP
Test substance::no dataother TS: purity: > 98 %Remark:Result:Initiation treatment: 3,2'-Dimethyl-4-aminobiphenyl (DMAB)
was subcutaneously injected at a dosage level of 50 mg/kg bw
once a week for 10 weeks.
Promotion treatment: 20 animals aged 4 weeks and 30 animals
aged 54 weeks received 2,6-di-tert-butyl-p-cresol (BC) withthe diet starting 1 week prior to the first initiation
treatment.
Control groups: 20 animals aged 4, and 30 animals aged 54
weeks served as the young and old control group,
respectively.
Low number of animals per dose group.
Significantly increased incidence in urinary bladder tumours
in both young (19/20) and old (29/30) group (no urinary
bladder tumours were present in either young or old control
group).
Induction of liver foci in both age groups was inhibited(3/20 and 4/30 in comparison to 19/20 and 18/30 in thecorresponding young and old control groups, respectively)
and the incidence of hyperplastic nodules in the liver was
decreased in the old group (0/30 as compared with 7/30 in
the control group).
Induction of pancreatic acinar cell foci was also inhibited(1/20 and 3/30 in comparison to 9/20 and 15/30 in thecontrol groups for young and old animals, respectively).
Tumour development in the small and large intestines,
prostate, preputial glands, skin/subcutis and ear duct
showed no modification by BC. No ageing effect was evident.
Moreover, DMAB-DNA adduct formation in rats given a single
administration of DMAB after 1-week treatment with BC(evaluated by an additional conducted enzyme-linked immunosorbent assay and immunohistochemical staining) correlated
well with tumourigenesis in the urinary bladder, liver andpancreas.

26.03.2003(144)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
32 weeksdaily
none
1 % in diet (ca. 500 mg/kg bw day)Remark:Initiation treatment: Successive administrations of 0.05 %N-butyl-N-(4-hydroxybutyl)-nitrosamine, 0.2 % N-bis(2hydroxypropyl)nitrosamine and 0.2 % N-ethyl-N-hydroxyethylnitrosamine each for 1 week in the drinking water with 3-day
exposition-free intervals between the treatment periods.
Promotion treatment: 14 animals received 2,6-di-tert-butylp-cresol starting after a 3-day exposure-free interval

134

other: concurrent, no initiation treatment or no promotion treatment
other: Wide-Spectrum Initiation/Promotion Assay1989
no datano data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

Result2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

subsequent to the last initiation treatment.
Control groups: Initiation treatments only (20 animals),promotion treatment without initiation treatment (13animals).
No informations on purity of TS.
Bladder: significantly increased incidences and numbers ofpapillary or nodulary hyperplasias (12/14; 3.43 +- 2.14/10
cm basement membrane) and of papillomas (6/14; 0.59+- 0.75/10 cm basement membrane) as compared with carcinogentreated control (3/20; 0.19 +- 0.51/10 cm basement
membrane or 1/20; 0.05 +- 0.21/10 cm basement membrane).
Thyroid: significantly increased incidence of adenomas(12/14) as compared with the carcinogen treated control(7/20).
Liver: significant decrease in the incidence (7/14) andnumber (0.37 +- 0.42/cm2) but not in the area/cm2 of hyperplastic foci (carcinogen treated control: 19/20; 2.12+- 1.48/cm2).
The following parameters were not modified by 2,6-di-tertbutyl-p-cresol: incidences of adenomas and adenocarcinomas
in the lung; incidences of adenomas in the renal cortex andof papillary or nodulary hyperplasia in the renal pelvis.

26.03.2003(145)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group

:
:
:
:
:
:
:
:
:
:

MethodYear
GLP
Test substance

:
:
:
:

Remark:Result:rat
male
Fischer 344/DuCrj
oral feed
30 weeksdaily
none
1 % in diet (ca. 500 mg/kg bw day)other: concurrent, no initiation treatment or no
modification
treatment
other: Lung and Thyroid Carcinogenesis Modification Assay1990
no datano data
Initiation treatment: N-bis(2-hydroxypropyl)nitrosamine(DHPN) was administered at a concentration level of 0.1 % in
the drinking water for 2 weeks.
Modification treatment: 20 animals received 2,6-di-tertbutyl-p-cresol (BC) after termination of initiation
treatment.
Control groups: Initiation treatment only (20 animals),
BC administration without previous initiation (10 animals).
No informations on purity of TS.
Significantly decreased lung carcinomas (1/20) in comparison
with carcinogen treated control (9/20); incidences of DHPNinduced lung adenomas were not affected; quantitative
analysis of adenomas and carcinomas (number and area of
lesions per unit area of lung section) revealed obvious
prevention of DHPN-induced carcinogenesis.
In contrast, DHPN-induced thyroid tumourigenesis was
significantly enhanced (14/20 in comparison with 5/20 in thecontrol group).

26.03.2003
Species

(146)
:

rat

UNEP PUBLICATIONS

135

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

male
Wistaroral feed3 - 22 weeks
daily
none
0.5 % in diet (ca. 250 mg/kg bw day)Remark:Result:Initiation treatment: Single i.p. injection of 200 mg
diethylnitrosamine/kg bw.
Selection treatment (starting 2 weeks after the initiation
treatment): rats were fed a 0.03 % 2-acetylaminofluorene(2-AAF) containing diet for a 2-week period, a single
intragastric dose of 2 ml CCl4/kg bw was administered after
1 week of 2-AAF feeding.
Promotion treatment: Starting 1 week after termination of
selection treatment.
3 animals/group were sacrificed, respectively after 3, 6, 14
or 22 weeks of administration of the 2,6-di-tert-butyl-pcresol (BC) diet.
Parameters: Interphase nuclear DNA content in hepatocellularlesions (foci or nodules) and lesion-surrounding area.
No informations on purity of TS.
BC treated animals, which developed no liver carcinoma
within the considered timespan, showed a clearly increased
amount of 2N nuclei in the precancerous lesions only after
14 weeks of treatment. It seems that there is a positive
correlation between the outgrowth of putative preneoplastic
foci and nodules in rat liver and an increase of diploid
nuclei in these lesions.

other: concurrent, no promotion treatment
other: Initiation-Selection/Promotion Assay1983
no datano data

26.03.2003(147)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
29 weeks and 4 days
daily
none
0.7 % in diet (ca. 350 mg/kg bw day)Remark:Initiation treatments: Single intragastric administration of
100 mg N-methyl-N'-nitro-N-nitrosoguanidine/kg bw, a single
intragastric administration of 750 mg N-ethyl-N-hydroxyethylnitrosamine/kg bw, two subcutaneous injections of
0.5 mg N-methylbenzylnitrosamine/kg bw once per 6 days, four
subcutaneous injections of 40 mg 1,2-dimethylhydrazine/kg bw
once every 3 - 4 days. At the same time the rats were given

136

other: concurrent, no initiation and/or promotion treatment
other: Multi-Organ Initiation/Promotion Assay
1993
no dataother TS: purity: > 98.0 %UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Result:Source26.03.2003:

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

Remark:Result:0.1 % N-dibutylnitrosamine for 4 weeks and then 0.1 % 2,2'dihydroxy-di-n-propylnitrosamine for 2 weeks in the drinking
water (for a total carcinogen exposure period of 6 weeks).
Promotion treatment: Starting 3 days after termination of
initiation treatment period.
Animals were sacrificed at the end of week 36 of the wholestudy.
Low number of animals per group.
Thyroid gland: significantly increased incidence of hyperplasia (13/17) and adenoma or carcinoma (6/18) as comparedto carcinogen treated control (0/18 each).
Colon: significantly decreased incidence (0/18) andmultiplicity (0) of colon adenocarcinoma as compared tocarcinogen treated control (10/18; 0.67 +- 0.69/animal).
Liver: significantly decreased multiplicity of atypicalrenal tubules (0.70 +- 0.58) as compared to carcinogentreated control (1.81 +- 0.84) and significantly decreased
incidence (1/18) and multiplicity (0.03 +- 0.11) of renal
cell tumours as compared to carcinogen treated control(8/18; 0.24 +- 0.31).
No significant modification of carcinogen induced lesions in
the following organs: tongue, esophagus, forestomach,glandular stomach, duodenum, small intestine, liver, lung,urinary bladder.
Shell
(148)
rat
male
Fischer 344
oral feed
76 weeks
none
100, 300, 1000, 3000 and 6000 ppm (ca. 7.5, 23, 75, 225 and 450 mg/kg
bw day)yes, concurrent no treatment
other: see remark field1990
no dataother TS: purity: > 99 %The study was not designed as definitive chronic bioassay.
21 rats/dose and 36 control rats; the diets were prepared
every 4 weeks and stored at 4°C until use (no analytical
data available); interim kill at 12, 36 and 48 weeks of 4
randomly selected animals; observations of pathology: To
demonstrate a deficiency in iron storage in cells of alteredhepatocellular foci, rats were iron-loaded with sc
injections of 12.5 mg elemental iron/100 g body weight in
the inguinal regions, alternating sides 3 times/week for 2
weeks prior to killing. Complete autopsies livers were
performed on all animals. At autospsy, livers were weighedand slices from each lobe were taken and fixed in 10%
neutral buffered formalin. Sections were stanined withhaematoxylin and eosin and tested for iron to determine thepresence of iron storage-deficient lesions. Tumors andlesions of other organs were submitted for histology.
HISTOPATHOLOGICAL EXAMINATION (liver):- Hepatocellular carcinomas: none observed in any group

UNEP PUBLICATIONS

137

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004- Hepatocellular adenomas: detected in all groups, including
controls: 17% (control), 14% (100 ppm), 29% (300 ppm), 29%(1000 ppm), 14% (3000 ppm), 33% (6000 ppm); notreatment-related trend in incidence(see also Chapter 5.4 for clinical results andhistopathological examinations of altered hepatic foci)(2) valid with restrictions
Critical study for SIDS endpoint

Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male
Fischer 344
oral feed
110 weeks

Remark:Result:Reliability
Flag
26.03.2003::

27 rats were fed the basal diet (control) and 27 rats
recieved 12000 ppm BHT in the diet. All animals were killed
at 110 weeks.
Observations of pathology: To demonstrate a deficiency iniron storage in cells of altered hepatocellular foci, rats
were iron-loaded with sc injections of 12.5 mg elemental
iron/100 g body weight in the inguinal regions, alternatingsides 3 times/week for 2 weeks prior to killing. Complete
autopsies livers were performed on all animals. At autospsy,
livers were weighed and slices from each lobe were taken and
fixed in 10% neutral buffered formalin. Sections were
stanined with haematoxylin and eosin and tested for iron todetermine the presence of iron storage-deficient lesions.
Tumors and lesions of other organs were submitted forhistology.
HISTOPATHOLOGICAL EXAMINATION (liver):- Hepatocellular carcinomas: none observed- Hepatocellular adenomas: incidence lower in treated group(13%) compared to control (36%)(see also Chapter 5.4 for clinical results andhistopathological examinations of altered hepatic foci)(2) valid with restrictions
Critical study for SIDS endpoint

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
Method

:
:
:
:
:
:
:
:
:
:
:

138

(78)

none
12000 ppm (ca. 900 mg/kg bw day)yes, concurrent no treatment
other: see remark field1990
no dataother TS: purity: > 99 %

(78)
rat
male/female
Wistaroral feed
104 weeksnone
0.25 or 1% in diet
negative
yes, concurrent no treatment

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Year
GLP
Test substance

:
:
:

1981
no
other TS: pure (for additive use)

Result:MORTALITY AND TIME TO DEATH: significant increase after 96
weeks in high-dose group of males (40% vs. 68% in control attermination of study)
CLINICAL SIGNS: not reported
BODY WEIGHT GAIN: significantly reduced in high-dose males
up to week 60 and in high-dose females for most of the study
FOOD CONSUMPTION: similar for treated and control animals
CLINICAL CHEMISTRY: dose-related changes in serum
triglyceride (reduction) and GGT (increase) in treated males
and in total blood cholesterol (increase) in treated females
HAEMATOLOGY: increase in red blood cell count in treatedfemales (both dose groups, but not dose-related)
ORGAN WEIGHTS: increased mean absolute and relative liver
weights in all treated animals; decreased absolute andrelative spleen weights in the treated females

Test condition:

Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

GROSS PATHOLOGY: no significant morphological changes in theliver
HISTOPATHOLOGY: a variety of tumours were observed, but theincidence of tumours was not significantly higher in treatedas compared to control animals
TEST ORGANISMS- Age: 7 weeks- Weight at study initiation: 100-200 g- Number of animals: 57/sex/group; 36/sex in control group
CLINICAL OBSERVATIONS:- Body weight: weekly- Food consumption: at regular intervals- Haematology: at week 104- Clinical chemistry: at week 104
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC ANDMICROSCOPIC):- Macroscopic: liver, spleen, kidney- Microscopic: liver, pancreas, mammary gland, uterus,
pituitary gland, adrenal gland, other selected organs andtissues
STATISTICAL METHODS: Student's t-test and chi-square test(2) valid with restrictions
Critical study for SIDS endpoint
(149)
mouse
male/female
B6C3F1oral feed
104 weeks
daily
16 weeks
1 and 2 % in diet (males: 1640 and 3480 mg/kg bw day;and 4130 mg/kg bw day)females: 1750

yes, concurrent no treatment
other: Carcinogenicity Study
1988
no dataother TS

UNEP PUBLICATIONS

139

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Result:50 mice/sex/dose and control group; results concerning
long-term toxicity except for carcinogenic effects are
depicted in chapter 5.4.
The following incidences of hepatocellular lesions were
found:
males
females___________________________________________________________
dose [%]
0
1
2
0
1
2
no. of mice

32

42

47

41

no. of mice with
hepatocellulartumours12(38) 26(62) 31(66)4440

7(17) 8(18) 2(5)no. of mice with- singleadenoma
3 (9) 11(26) 8(17) 4(10) 7(16) 2(5)- multiple
adenomas
2 (6) 4(10) 15(32)* 1 (2) 00- adenomas andcarcinomas 1 (3) 1 (2) 2 (4) 000- carcinomas 6(19) 10(24) 6(13) 2 (5) 1 (2) 0in parentheses: incidences in %* significantly different from 0 % group at p < 0.01

Test substance:

Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
Method
Year

:
:
:
:
:
:
:
:
:
:
:
:

140

The incidence of hepatocellular adenomas, but not
carcinomas, was increased significantly in males at the 2 %concentration level. The 2 % females showed less hepatocellular tumours than the control females. No other tumour
rate was increased.
The liver tumour incidence in control animals was elevated in comparison
to earlier studies performed in the same laboratory as well as compared tohistorical control data published by NTP (see page 54 of publication).
Survival:- Males: dose-related increase; survival rates at week 104:
74% (high dose), 64% (low dose), 40% (control)- Females: no differences until week 88; survival rates atweek 104: 89% (high dose), 81% (low dose), 58% (control)
purity: 96 % (an actual level of almost 50 % of the initial
2,6-di-tert-butyl-p-cresol content was determined in thefeed pellets, the distribution of the test substance in thepellets seemed to be inhomogenous)(2) valid with restrictions
Critical study for SIDS endpoint
(87)
mouse
male/female
C3H
oral feed
10 months
daily
none
0.05 and 0.5 % in diet (ca. 39 and 390 mg/kg bw day)yes, concurrent no treatment
other: Carcinogenicity Study
1986

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

GLP
Test substance::no datano data

Remark:Result:Body weight gain was reduced in males at the end of study;
number of animals:
control diet:
40 males, 39 (?) females;
laboratory chow:
38 males, 28 (?) females;
0.05 % test substance: 26 males, 29 (?) females;
0.5 % test substance: 36 males, 46 (?) females.
No informations on purity of TS.
The feeding of 0.5 or 0.05 % test substance significantly
increased the incidence of liver tumours in males whencompared to the animals kept on the 2,6-di-tert-butyl-pcresol-free control diet. The lower concentration of test
substance was apparently more effective in enhancing liver
tumour development than was the higher concentration,although the difference between the two groups was not
significant. The incidence of spontaneously developing lungtumours was not modified by the 2,6-di-tert-butyl-p-cresol
treatment.
dose groupsex
tumour incidence
liver
lung
____________________________________________________________
control dietmale
2/37 (5 %) 2/37 (5 %)(2,6-di-tert-butyl- female 0/39 (0 %) 4/39 (10 %)p-cresol free)standard laboratory male
7/38 (18 %) 2/38 (5 %)chowfemale 2/28 (7 %) 4/28 (14 %)0.05 % 2,6-di-tert- malebutyl-p-cresol
female15/26 (58 %)sS 5/26 (19 %)1/29 (3 %) 3/29 (10 %)0.5 % 2,6-di-tert- male 10/36 (28 %)s 5/36 (14 %)butyl-p-cresol
female 2/46 (4 %) 9/46 (20 %)s significant increase compared to the control diet group(p < 0.05)
S significant increase compared to the laboratory chowgroup (p < 0.05)
The validity of these results is low because of inexact
descriptions, the small number of animals, the shorttreatment period and the high incidence of spontaneously
developing tumours.
The authors concluded that 2,6-di-tert-butyl-p-cresol
increases the incidence of spontaneously developing tumours,
and therefore, acts as tumour promotor in mouse liver.
19.11.2001
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group

(150)
:
:
:
:
:
:
:
:
:
:

mouse
male/female
C3H
oral feed
1 monthdaily
9 months
0.5 % in diet (ca. 390 mg/kg bw day)yes, concurrent no treatment

UNEP PUBLICATIONS141

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

MethodYear
GLP
Test substance

:
:
:
:

other: Carcinogenicity Study
1986
no datano data

Remark:Result19.11.2001:

Animals were fed with laboratory chow during the postexposure period; body weight gain was reduced in males atthe end of study. Liver and lung were evaluated for tumours.
17 - 38 males/dose and control groups ("control diet" orlaboratory chow), 17 or 38 females/dose and laboratory chow
control group; data concerning the number of animals wereconflicting in the publication.
No informations on purity of TS.
Treatment was without significant effect.

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:

mouse
male/female
B6C3F1oral feed
107 - 108 weeks
daily
none
3000 and 6000 ppm (ca. 450 and 900 mg/kg bw day)Remark:Result:Reliability
Flag
26.03.2003::

Body weight gain was reduced in a dose-related manner;survival was not affected by test substance treatment;
50 mice/sex and dose group, control: 20 mice/sex.
The incidence of alveolar/bronchiolar carcinomas or adenomas
was significantly increased in the female mice in the lowdose group, but the incidences were not significantly doserelated (control: 1/20, low dose: 16/46, high dose: 7/50).
Thus, these lung tumours in the females cannot clearly be
related to the administration of 2,6-di-tert-butyl-p-cresol.
Significant results in the negative direction were observedin the incidence of tumours of the liver in male mice and in
the incidence of sarcomas of multiple organs in female mice.
(1) valid without restriction
Critical study for SIDS endpoint

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
Method
Year
GLP

:
:
:
:
:
:
:
:
:
:
:
:
:

142

(150)

yes, concurrent vehicle
other: Carcinogenicity Study
1979
no dataother TS: purity: 99.9 %

(133)
mouse
male/female
Balb/coral feed
18 months
daily
none
0.75 % in diet (ca. 1125 mg/kg bw day)yes, concurrent no treatment
other: Carcinogenicity Study
1978
no data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Test substance:no data

Remark:Result:Survival was increased significantly in males;
100 males/dose and control group; 50 females/dose andcontrol group.
No informations on purity of TS.
The incidence of leukemias, tumours of liver or forestomach
as well as forestomach hyperplasia was not increased by the
2,6-di-tert-butyl-p-cresol treatment.

26.03.2003(151)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group

:
:
:
:
:
:
:
:
:
:

MethodYear
GLP
Test substance

:
:
:
:

Remark:Result

:

mouse
male
B6C3F1oral feed
24 weeksdailynone
5000 ppm in diet (ca. 750 mg/kg bw day)other: concurrent, initiation treatment only, no treatment at all or positive
promotion control after initiation
other: Liver Carcinogenesis Promotion Assay1982
no dataother TS: purity: > 99 %Initiation treatment: Diethylnitrosamine (DENA) was intraperitoneally injected at dosages of 100 or 200 umol/kg bw
per administration once a week for 10 weeks.
Promotion treatment: Starting after a 4-week exposure-freerecovery interval subsequent to the last initiation
treatment 40 animals/initiation dose group received 2,6-ditert-butyl-p-cresol until sacrifice at week 38 of the wholestudy.
Control groups: No treatment at all (20 animals), initiation
treatments only (45 animals/DENA dose group), positive promotion with 500 ppm phenobarbital in the diet (40 animals/
DENA dose group).
Parameters: Hepatocellular foci, adenomas and hepatocellular
carcinomas were diagnosed.
Low number of animals in untreated control group.
The incidence and the multiplicity of both altered liverfoci and adenomas or carcinomas were not affected by 2,6-ditert-butyl-p-cresol administration.

26.03.2003(152)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
Method
Year

:
:
:
:

mouse
male/female
B6C3F1oral feed
96 weeksdaily
8 weeks
200, 1000 and 5000 ppm in diet (ca. 30 - 750 mg/kg bw day; actual levels:
200, 800 and 4000 ppm (ca. 30-600 mg/kg bw day)yes, concurrent no treatment
other: Carcinogenicity Study
1982

UNEP PUBLICATIONS

143

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

GLP
Test substance::no dataother TS: food additive grade

Remark:Result:Reliability
Flag
26.03.2003::

Females (1000 and 5000 ppm) and males (5000 ppm) showed
reduced weight gain. Neither survival rates nor foodconsumption differed between experimental and control
groups. No significant changes were found in the haematological examiniations or serum and urine analyses;
51 - 52 mice/sex and dose group, control group: 50 mice/sex.
Tumours were found in many organs, especially in the lungs,
liver, lymph nodes and spleen, in both the experimental andcontrol groups, but none were related to the2,6-di-tert-butyl-p-cresol treatment. Thus, this experiment
provided no evidence of 2,6-di-tert-butyl-p-cresol
carcinogenicity in mice.
(1) valid without restriction
Critical study for SIDS endpoint

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses

:
:
:
:
:
:
:
:

ResultControl group
MethodYear
GLP
Test substance

:
:
:
:
:
:

RemarkResult

:
:

(153)
mouse
male/female
other: CF1
oral feed
2 yearsdaily
none
1000, 2500 and 5000 ppm; during the first 4 weeks alltreatment groups
received 1000 ppm (ca. 150, 375 and 750 mg/kg bw day; 150 mg/kg bw
day during the first 4 weeks)yes, concurrent vehicle
other: Carcinogenicty Study
1992
no dataother TS: purity: > 99.5 %High spontaneous incidence of lung tumors.
No compound-related increase in liver cell carcinomas were
found, but an exposure-related increase in the incidence oflung neoplasia was established.
The authors stated, that it had not been possible to clear
up the role of 2,6-di-tert-butyl-p-cresol as a carcinogen inmice in this study, because of the relatively highbackground incidence of lung tumours.

26.03.2003
Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Post exposure period
Doses
ResultControl group
MethodYear
GLP
Test substance

144

(154)
:
:
:
:
:
:
:
:
:
:
:
:
:
:

mouse
male/female
B6C3F1oral feed
107-108 weeks
daily
none
3000 and 6000 (ca. 450 and 900 mg/kg bw day)yes, concurrent no treatment
other: Carcinogenicity Study
1979
no dataother TS: purity: 99.9%

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Result:Reliability
Flag
26.03.2003

:
:

5.8.1

Body weight gain was reduced in dosed mice; survival was notaffected by test substance treatment;
50 mice/sex and dose group, control: 20 mice/sex.
The incidence of alveolar/bronchiolar carcinomas or adenomas
was significantly increased in the female mice in the lowdose group, but the incidences were not significantly doserelated (control: 1/20, low dose: 16/46, high dose: 7/50).
Thus, these lung tumours in the females cannot clearly be
related to the administration of 2,6-di-tert-butyl-p-cresol.
Significant results in the negative direction were observedin the incidence of tumours of the liver in male mice and in
the incidence of sarcomas of multiple organs in female mice.
(1) valid without restriction
Critical study for SIDS endpoint(133)

TOXICITY TO FERTILITY

Type
Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:
:

One generation study
ratmale/female
Wistaroral feed
F0, females: during premating, mating, gestation, and lactation; F0, males:
during premating and mating;F1: until weaning
daily

Frequency of treatm.
:
Premating exposure periodMale: 5 - 7 weeks
Female : 5 - 7 weeks
Duration of test
: until pups reached ca. 8 weeks of age
No. of generation:
studies
Doses: 25, 100, 250, 500, 750 and 1000 mg/kg bw day
Control group: yes, concurrent vehicle
NOAEL parental: < 500 mg/kg bw
NOAEL F1 offspring: < 500 mg/kg bw
Method: other: Reproduction StudyYear: 1990GLP: no data
Test substance: no data
Remark:Result:26.03.2003
Type
Species
Sex
StrainRoute of admin.

:
:
:
:
:

no data concerning the number of animals. No informations on
purity of TS.
Apart from a slight reduction in mating success in rats
receiving the highest dose, no treatment-related effect ondams or pups was found up to the time of birth.
However, pups of dams initially receiving >= 500 mg/kg bw day showed amarked loss of weight at weaning and 4 weeks on, although being fed thecontrol diet. No pathological changes were observed in the pups, and dams
showed no signs of malnutrition. The authors suggested that the low weightgain of high dose-pups was due to metabolic competition (namely milk
production versus mixed function oxidase activity).
(155) (156) (157) (158)other: Dose-ranging study
ratmale/female
Wistarother: diet

UNEP PUBLICATIONS

145

OECD SIDS
5. TOXICITY
Exposure period

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

male: 5 weeks before mating; 21 day after birth
female: 5 weeks before mating; 21 day after birth(F= and the majority of F!)

Frequency of treatm.
:
Premating exposure period
Male:Female :Duration of test:
No. of generation:
studies
Doses: 500, 750 and 1000 mg/kg bw day
Control group: yes, concurrent no treatment
Method
:
Year
:
GLP
:
Test substance: other TS: purity: 99.9%
Remark:Result:26.03.2003
Type
Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:
:

post observation period: 4 wk (F1); limited study for
dose-finding.
Dams treated with doses of 500 mg/kg bw day upwards showed areduction in bw gain during lactation. Liver weight wasincreased at all dose levels; no histological changes in the livers of dams
killed at the estimated day 19 or 20 of
gestation; proliferation of the endoplasmic reticulum within hepatocytes atthe end of lactation in the 750 and 1000 mg/kg bw day dose-groups.There
was no significant difference in overall mating success between anygroups.The body weight of pubs did not return to normal following a return
to a control diet for 4 wk.
(85)
other: Three-generation study
mouse
male/female
other: Crj:CD-1
oral feed
F0 and F1: during premating, mating, gestation and lactation (ca. 11
weeks)
daily

Frequency of treatm.
:
Premating exposure periodMale: 4 weeks
Female : 4 weeks
Duration of test
: until postnatal day 21 of the F2 generation
No. of generation:
studies
Doses: 0.015, 0.045, 0.135 and 0.405 % in diet (ca. 22.5, 67.5, 202.5 and 607.5
mg/kg bw day; doses estimated based on approximative daily food intake
for mice)
Control group: yes, concurrent no treatment
NOAEL F1 offspring: .405 %NOAEL F2 offspring: .405 %Method: other: see remark fieldYear: 1993
GLP: no data
Test substance: no data
Remark146:

METHOD: No. of mice/sex/dose: 10; mating period: 5 days; M/Fratio per cage: 1/1; length of cohabitation: no data;
neurobehavioural procedure: The functional and behaviouraldevelopmental parameters were measured and scored for theindividual pups in the lactation period in F1 and F2

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
generations, and were analyzed on a whole-litter basis. The
measured parameters were as follows: surface righting on
postnatal day 4 and 7, negative geotaxis on PND 4 and 7,
cliff avoidance on PND 7, swimming behaviour (direction,
head angle, and limb movement) on PND 4 and 14, andolfactory orientation on PND 14. Open field activity of mice
was measured at 3 weeks of age in the F1 and F2 generations,both male and female. the apparatus used in this study was asquare white board, 30 x 30 cm, divided by black lines into
25 equal squares. Ambulation, rearing, 180° turn,defectation, urination, and preening were recorded for 3 minin the apparatus. In the F1 generation, the following
parameters were measured on postnatal (PND) 0: litter size,
litter weight, and sex ration (m/f); the pups were weighedon PND 0,4,7,14 and 21 in the lactation period; the pups
were removed from their dams at 4 weeks of age, and wereselected at random to continue treatment; the F1 animals
were mated at 9 weeks of age; in the F2 generation someparameter of the pups were measured identically to the F1generation from birth to weaning. For the F0 generation only
data on mortality are reported administration of BHT: nofurther information given.

Result:Doses estimated based on approximative daily food intake formice.
F0 generation:
MORTALITY: Two dams died during the second week of thelactation period; one dam in the 0.015% group and one in the
0.045% group.
F1 generation:
0.015%:MORTALITY: 1 dam died during 2nd week of lactation period
SURVIVAL INDEX (PND 21): 100% (control: 91.8%)
BODY WEIGHT: increased at PND 0,4 and 21
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: increased surface righting at
PND 7
0.045%:SURVIVAL INDEX (PND 21): 90.3% (control: 91.8%)
BODY WEIGHT: no effectNO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: reduced ambulation in male mice
0.135%:SURVIVAL INDEX (PND 21): 100% (control: 91.8%)
BODY WEIGHT: decreased at PND 14
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: no effect
0.405%:SURVIVAL INDEX (PND 21): 98.3% (control: 91.8%)

UNEP PUBLICATIONS

147

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
26.03.2003

:
:

Type
Species
Sex

:
:
:

148

BODY WEIGHT: decreased at PND 7, 14 and 21
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: no effect
F2 generation:
0.015%:SURVIVAL INDEX (PND 21): 100% (control: 100%)
BODY WEIGHT: increased at PND 0,4, 7, 14 and 21
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: reduced 180o turn (m)0.045%:SURVIVAL INDEX (PND 21): 99.1% (control: 100%)
BODY WEIGHT: no effectNO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: reduced 180o turn (m), reduced
ambulation in both sex
0.135%:SURVIVAL INDEX (PND 21): 99.1% (control: 100%)
BODY WEIGHT: decreased at PND 14
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: increased surface righting at
PND 4, reduced 180o turn (m)0.405%:SURVIVAL INDEX (PND 21): 99.1% (control: 100%)
BODY WEIGHT: decreased at PND 7, 14 and 21
NO. of LITTERS: no effectNO. of PUPS: no effect
LITTER SIZE: no effect
LITTER WEIGHT: no effect
SEX RATIO: no effect
NEUROBEHAVIOURAL PARAMETERS: increased negative geotaxis at
PND 4, reduced 180o turn (m)CONCLUSION:No effect on No. of litters, No. of pups, litter size,
litter weight and sex ratio in any dose group of F1 and F2
animals; no effect on neurobehavioural parameters in F1 andF2 generation; the body weight of pups was increased in the
0.015% group at birth and during lactation period for eachgeneration(2) valid with restrictions
Critical study for SIDS endpoint(159)
other: two generation carcinogenicity study
ratmale/female

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY
StrainRoute of admin.
Exposure period

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:::

Wistaroral feedmale: 14 weeks (F0); 141-144 weeks (F1)female: 20 weeks (F0); 141-144 weeks (F1)dailyFrequency of treatm.
:
Premating exposure periodMale: 13 weeks
Female : 13 weeks
Duration of test
: 144 weeks
No. of generation:
studies
Doses: nominal: 0,25,100 and 500 mg/kg bw day (F0); 0, 25, 100 and 250 mg/kg
bw day (F1)
Control group: yes, concurrent no treatment
Method: other: see remark
Year: 1986
GLP: no data
Test substance: other TS: purity: > 99.5 %Remark:Result:Reliability
Flag
26.03.2003::

METHOD: No. of rats/sex/dose: 60 (control); 40 (25 mg/kg bw day), 40(100 mg/kg bw day) and 100 (500 mg/kg bw day); mating period was
terminated within 1week; M/F ratio per cage: no data; length of
cohabitation: no data; The only data reported are: gestation rate, No. of
pups/litter and the body weight of pups at birth and at weaning
F0 generation:
No difference was found in food consumption between treatedand control rats; body weight gain of males and females was
reduced significantly from week 6 of treatment with 500
mg/kg bw day, persisting throughout the lifespan of the F0 rats.
Gestation rate was not affected by treatment (or evenslightly increased in the treated groups).
The Armitage-Cochran test for linear trend in proportions demonstrated
that the fraction of litters with ten or more pups decreased significantly with
BHT dose (P<0.001).
F1 generation:
At weaning F1 rats had significantly lower body weights than the controls,
the extend of the reduction being
dose-related, although food consumption was not reduced in
the treated groups. The effect was most pronounced in males.
500 mg/kg bw day: decreased body weight (m/f) at weaning; the fraction oflitters with ten or more pups decreased
100 mg/kg bw day: decreased body weight (m/f) at birth and at weaning
25 mg/kg bw day: no effects
Reproduction data for F0 rats fed BHT (in mg/kg bw day)
0 25 100 500
pups/litter (mean)
10.9 9.6 10.3 9.1*
pups/litter (standardized)
8.0 8.0 8.0 7.9
pups/litter (at weaning)
7.9 8.0 7.7 7.8
pup body weight° [g] (at birth) 5.9 5.9 5.7# 5.7
pup body weight° [g] (at weaning) 42.4 40.4# 39.7## 25.3##°) Average of mean pup weight/litter. Values marked with (*) or (#) show
statistically significant difference from the control: *: P<0.001 by the
Armitage-Cochran test for linear trend in proportions of litters with ten or
more pups; #: P<0.05 and ##: P<0.001 by Student's test.
The pathology findings (F1) including blood analysis andserum chemistry are presented in chapter 5.4 and5.7("Repeated Dose Toxicity" and "Carcinogenicity").
(2) valid with restrictions
Critical study for SIDS endpoint(79)

UNEP PUBLICATIONS

149

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type

:

Species
Sex
StrainRoute of admin.
Exposure period

:
:
:
:
:

Frequency of treatm.

:

other: two generation study with emphasis on hepatocellular changes in F1
generation
rat
male/female
Wistarother: dietmale: 5 weeks (P); 4 weeks (F1), 6, 11, 16
and 22 months (F1)female: 8 weeks (P)daily (during the period of mating, food pots were removed when male andfemales were mated)

Premating exposure periodMale: 3 weeks
Female : 3 weeks
Duration of test
: 22 months
No. of generation:
studies
Doses: nominal: 0,25,100 and 500 mg/kg bw day (P); 0, 25, 100 and 250 mg/kg bw
day (F1)
Control group: yes, concurrent no treatment
Method: other: see remark
Year: 1994GLP: yes
Test substance: other TS: purity: 99.96%
Remark:Result:150

NOAEL PARENTAL:
The NOEL for clinical signs during premating and matingphases, for both males and females, was 500 mg/kg bw day.
The NOEL for effects on body weight during premating andmating phases was 500 mg/kg bw day for the females and 100 mg/kg bw
day for the males.
The NOEL for maternal clinical signs and for effects onmaternal body weight during gestationphase was 500 mg/kg bw day. The
NOEL for maternal clinical signs and for effects on maternal body weight
and food consumption during thelactation phase was 500 mg/kg bw day.
NOAEL F1 OFFSPRING:
The NOEL for pup clinical signs were 500 mg/kg bw day; the NOEL for pup
body weight during lactation phase were 100 mg/kg bw day.
METHOD: premating exposure period for males (7/dose) andfemales (50/dose): 3 weeks; mating exposure period for males(6/dose) and females (48/dose): 2 weeks; M/F ratio per cage:
1/8; length of cohabitation: 15 hours/day; number of animals
allocated for each scheduled autopsy: 20 days gestation: 5
pregnat females/dose, 21 days after parturition: 5mothers/dose and 20 pups/dose, 4 weeks after weaning: 5 malepups/dose, 6 months after weaning: 5 male pups/dose; 11months after weaning: 8-10 male pups/dose, 16 months after
weaning: 9-13 male pups/dose, 22 months after weaning: 10-19
male pups, administration of BHT: the amount of BHT
incorporated initially per unit weight of diet wascalculated from the food consumption measured during
acclimatisation and from normal growth rate of this strainof rats; throughout pregnancy and lactation no effort wasmade to adjust dietry BHT content in line with body weightgain during this time
There were no differences in mating success. Pregnancy
proceeded normally in all groups. There was no alteration in
numbers of resorption sites. No statistically significant
change was seen in the number of foetuses/dams. The numberof pups per litter did not differ. There was a trend to an

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

Reliability
Flag
26.03.2003
5.8.2

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004::

increase in the number of pups found dead or dying soonafter birth with increase in dose but the actual number ofdeaths in affected litters was not influenced by treatment
with BHT.
The total litter weight was significantly decreased for dams
treated with the high dose of BHT. The weight gain of pups
from dams receiving the highest dose of BHT was
consistently less than that of control pups or pups of dams
receiving lower doses of BHT. The development was retardedin the high dose group.
The pathology findings (P and F1), including
liver-biochemistry, organ weights, gross and microscopic
evaluations are presented in chapter 5.4 (Repeated DoseToxicity").
(1) valid without restriction
Critical study for SIDS endpoint(85) (86)

DEVELOPMENTAL TOXICITY/TERATOGENICITY

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

rat
femaleSprague-Dawley
gavage7th to 17th day of gestation
daily
until day 20 of gestation
100, 200, 300 and 400 mg/kg bw dayother: no data
other: no data
1993
no dataother TS: BHT (no further information) in corn oil

RemarkResult::Reliability
Flag
26.03.2003::

only abstract
Pregnant performance and fetal developments were notaffected; no significant differences were detected inmaternal body weight gains and food intakes; a dose related
increase in relative organ weight of liver at high doses; no significant fetal
abnormalities in external and visceralobservations; on skeletal examination sternebral retardation in BHT 300
mg/kg bw day treated group were observed without dose dependence(4) not assignable
Critical study for SIDS endpoint(160)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

rat
femaleWistargavagedays 7 to 17 of pregnancy
daily
until day 20 of gestation
0, 93.5, 187 and 375 mg/kg bw dayother: no data
other: see remark field1990
no datano data

UNEP PUBLICATIONS

151

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Result:Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
NOAEL maternal tox.
NOAEL teratogen.
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

mouse
femaleother: JCL-ICR
gavage7th to 13th day of gestation
once a day
until the 18th day of gestation
70, 240 and 800 mg/kg bw dayother: yes, concurrent vehicle and concurrent untreated= 800 mg/kg bw= 800 mg/kg bw
other: see remark field

Remark:BHT was dissoved in olive oil and was administered at a rate of 10 ml/kg
bw dayAge at study initiation: 8-13 week old
Number of animals per dose and vehicle control: 26
Number of animals in untreated control: 30
Mating: After keeping a pair of male and female mice
together overnight, the female was examined in the nextmorning for the presence of vaginal plug. The mice with plugwere considered as pregnant animal. The day where female
mouse had virginal plug was designed as gestation day 0.
Body weight were measured everyday with the observation ofgeneral condition of the animal. The mice were sacrificed on
18th day of gestation by ether anesthetization. Immediately
after sacrifice, abdomen of the dam was opened, then thenumber of implantation sites, corpus luteum absorbed
embryos, dead or alive fetuses were counted. The alive
fetuses were examined for their body weights, sex andexternal malformation. Major organs were weighed and theabnormality was observed grossly. Five dams were chosen at152

abstract, figures and tables in English
METHOD: Number of animals per dose: 24 (control); 20 (93.5
and 187 mg/kg bw day); 22 (375 mg/kg bw day)
MATERNAL PARAMETERS assessed: clinical signs, body weight,
food consumption and mortality;
REPRODUCTIVE PARAMETERS assessed: number of corpora lutea,
number of implantation, number of live fetuses and sex ratio
FETAL PARAMETERS assessed: body weight; postnatal survival;
external abnormalities; visceral and skeletal abnormality
In the dams at the two higher doses of 187 and 375 mg/kg bw day, toxic
signs such as hair fluffing and diarrhoea wereobserved, and their body weight gain and food consumption
were suppressed. Two dams, which showed marked diarrhoea inthe highest dose group, died. However, there was no evidence of fetal
malformation attributable to treatment with the compound in any of thedose groups treated, although a slight increase in fetal death was found in
the highest dose group.
It is concluded that 2,2'-methylenebis (4-methyl-6-tertbutylphenol) has a weak lethal effect on fetal development
but not a teratogenic effect in the rat.
(4) not assignable
Critical study for SIDS endpoint(161)

no dataother TS: food additive grade

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
random and their alive fetuses were fixed with Bouin's
fixative for observation of internal abnormalities. The
remaining alive fetuses were fixed in 95% ethanol, then were
stained with alizarin red S for examination of skeletal
abnormalities.
MATERNAL PARAMETERS assessed: behavior; body weight;
mortality; organ weights (liver, heart, spleen, kidneys,lung, adrenals and ovaries);
REPRODUCTIVE PARAMETERS assessed: gestation rate; number ofcorpora lutea, number of implantation and sex ratio
FETAL PARAMETERS assessed: body weight; postnatal survival;
external abnormalities; skeletal deformity and abnormality
800 mg/kg bw day:
MATERNAL PARAMETER: increased spleen weight; decreased kidneyweight (compared to the untreated control animals)
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects
240 mg/kg bw day:
MATERNAL PARAMETER: no effects
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects
70 mg/kg bw day:
MATERNAL PARAMETER: no effects
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects(2) valid with restrictions
Critical study for SIDS endpoint(115)

Result:Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
NOAEL maternal tox.
NOAEL teratogen.
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

mouse
femaleother: JCL-ICR
gavage9th day of gestation
single administration
until the 18th day of gestation
1200 and 1800 mg/kg bw
yes, concurrent no treatment< 1200 mg/kg bw
1800 mg/kg bw
other: see remark field

Remark:BHT was dissoved in olive oil and was administered at a rateof 10 ml/kg bw day
Age at study initiation: 8-13 week old
Number of animals per dose: 15
Number of animals untreated control: 19
Mating: After keeping a pair of male and female mice
together overnight, the female was examined in the nextmorning for the presence of vaginal plug. The mice with plugwere considered as pregnant animal. The day where female
mouse had virginal plug was designed as gestation day 0.
Body weight were measured everyday with the observation ofgeneral condition of the animal. The mice were sacrificed on
18th day of gestation by ether anesthetization. Immediately
after sacrifice, abdomen of the dam was opened, then thenumber of implantation sites, corpus luteum absorbed
embryos, dead or alive fetuses were counted. The alive

no dataother TS: food additive grade

UNEP PUBLICATIONS

153

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
fetuses were examined for their body weights, sex andexternal malformation. Major organs were weighed and theabnormality was observed grossly. Five dams were chosen at
random and their alive fetuses were fixed with Bouin's
fixative for observation of internal abnormalities. The
remaining alive fetuses were fixed in 95% ethanol, then were
stained with alizarin red S for examination of skeletal
abnormalities.
MATERNAL PARAMETERS assessed: behavior; body weight;
mortality; organ weights (liver, heart, spleen, kidneys,lung, adrenals andovaries);
REPRODUCTIVE PARAMETERS assessed: gestation rate; number ofcorpora lutea, number of implantation and sex ratio
FETAL PARAMETERS assessed: body weight; postnatal survival;
external abnormalities; skeletal deformity and abnormality
1800 mg/kg bw:
MATERNAL PARAMETER: 5/20 died (11th day 3; 14th day 1 and15th day 1), increased lung and spleen weights
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: delay of progression of ossification
1200 mg/kg bw:
MATERNAL PARAMETER: 2/20 died (11th day 1 and 15th day 1),
increased lung weight
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: delay of progression of ossification(2) valid with restrictions
Critical study for SIDS endpoint(115)

Result:Reliability
Flag
26.03.2003::

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

rat
male/female
other: Norway Hooded
oral feed
5 months

Result:

Test condition:

Reliability:

No differences between teratment groups regarding number ofpups born and weaned and on the total weight of litter at 21days. Anophthalmia in 3 of 30 litters; number ofanophthalmic young to the total number in these threelitters: 1/11, 1/11, 8/11. With the small number it was notpossible to trace the cause of this effect to either genetic
or dietary factors. In a separate experiment, the mating ofanophthalmic male and female rats failed to produce
anophthalmic young even at a BHT concentration of 0.5% in
the diet. Anophthalmia was not observed in any otherexperimental animals used by the authors.
BHT was fed to male and female rats (16/group) for 5 or 6months. Reproductive studies were carried out at the 4thmonth followed by killing at six months (insufficient
documentation).
(3) invalid
Method with major limitations, e.g. limited number ofanimals ; effect of high lard intake unclear; insufficient154

6 months
0.1 or 0.5% in diets containing 10 or 20% lard supplement
yes, concurrent vehicle
other: reproductive study
1959
nono data

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
documentation

23.07.2001(162)

Species
Sex
StrainRoute of admin.
Exposure period
Frequency of treatm.
Duration of test
Doses
Control group
MethodYear
GLP
Test substance

:
:
:
:
:
:
:
:
:
:
:
:
:

mouse
femaleCD-1
oral feeddays 17 to 20 of pregnancy
daily
approx. 2-3 days
0.75 %

Remark:Result:

The ability of BHT to counteract the transplacentallyinduced retinal degeneration caused by
N-methyl-N-nitrosourea (MNU) was tested. For this purpose a
BHT-fed control (2 dams) was examined. Weanlings wereallowed to eat the the mixture ad libitum until they were
killed. Thus the offspring were exposed to BHT pre- andpost-natally.
No informations on purity of TS.
The retinas of BHT-fed control animals demonstrated sporadic
moprphologic changes in the form of circular configurations composed toganglion cells, arcades of nuclear and plexiform layers.
(163)

26.03.2003

other: see remark field1988
no datano data

5.8.3

TOXICITY TO REPRODUCTION, OTHER STUDIES

5.9

SPECIFIC INVESTIGATIONS

5.10

EXPOSURE EXPERIENCE

Remark:Source28.04.1994:

After a single oral dose of 0.5 mg/kg bw in man and of 0.25
mg/kg bw in woman a mean maximum of 75 ng 2,6-di-tert-butylp-cresol (BC)/ml was found in blood plasma (reached between
40 and 90 hours after administration). Large variations wereobserved between individual plasma curves.
Excretion of the applied dose within 4 days after ingestion:
0.2 +- 0.1 % as unconjugated BC acid with the urine(detection limit: 0.1 %), 2.6 +- 2.2 % as (un)conjugated BCacid with the urine, and non-detectable amounts of unchanged
test substance (detection limit: 3 - 5 %) with the faeces.
Almost the total amount was found on day 1 after ingestion.
7 male and 5 female volunteers each ingested a gelatine
capsule containing a 10 % solution of 2,6-di-tert-butyl-pcresol in corn oil.
Shell(164) (165)

UNEP PUBLICATIONS

155

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
30.10.2001::

Remark:2,6-di-tert-butyl-p-cresol was reported to be weakly
irritating and moderately sensitizing when patch-tested on>= 15 individuals in 1952: application to the skin led toslight erythema; second application after 14 days caused
erythema, edema and slight maceration.
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(67)

Despite of being in wide dispersive use for years, only a
few cases of skin sensitization due to 2,6-di-tert-butyl-pcresol as ingredient of various products are discussed.
Symptoms were contact dermatitis after dermal application of
2,6-di-tert-butyl-p-cresol and allergic reactions after oral
administration of a mixture of 2,6-di-tert-butyl-p-cresoland butylhydroxyanisol.
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(97) (69)

Poisoning case:A 22-year-old white woman ingested 4 g 2,6-di-tert-butyl-pcresol on an empty stomach as treatment for her genital
herpes simplex virus infection.
Later that evening she experienced severe epigastriccramping, general weakness, nausea, and vomiting, followedby dizziness, confusion, and a brief loss of conciousness.
She was admitted in a dehydrated condition with epigastricburning pain. The symptoms were resolved within a few daysof treatment (hydration, prochlorperazine, and antacids).
Poisoning cases(2) valid with restrictions
Critical study for SIDS endpoint(166)

Poisoning case:A 24-year-old woman ingested 80 g 2,6-di-tert-butyl-p-cresol
suspended in safflower oil on an empty stomach. Between 30
and 60 min later, she showed neurologic symptoms, which werecompletely reversible.
Poisoning cases(2) valid with restrictions
Critical study for SIDS endpoint(167)

Average concentration of 2,6-di-tert-butyl-p-cresol in
adipose tissue of men and women in Japan, USA, Canada, andGreat Britain (from various references):
Country
Japan

156

n

mg/kg
daily intake
in fat wet weight mg/person

11 m/7 f 0.10 0.0637
0.273(0.02 (0.0127(0 - 5.166)

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004- 0.18) - 0.115)
Great
Britain6 m/5 f 0.23 0.1530(0.01 (0.0070- 0.49) - 0.330)

1.0

USA

7 m/5 f 1.30 0.8700(0.34 (0.2300- 3.19) - 2.140)

2.0

Canada4 m/2 f 0.12
7.4
(0.07
- 0.19) _______________
in parentheses: minima and maxima
n: number of individuals
m: males; f: females
No significant difference was found between men and women.
(168) (169) (170) (171) (172)22.04.1994

Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
16.08.2001::

20 workers in the aerospace industry handling dielectric
fluids for electrodischarge machining and having developed
irritant contact dermatitis were patch tested with 2 %2,6-di-tert-butyl-p-cresol in petrolatum. The dielectric
fluids contained 0.2 % 2,6-di-tert-butyl-p-cresol as anadditive. All patch tests were negative.
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(70)

In an epidemiological study of 286 metalworkers exposed tometalworking fluids, no contact sensitization to 2,6-ditert-butyl-p-cresol was found when workers were patch tested
with 2 % in petrolatum (application to the upper back;fixing with "Fixomull" tape; contact period: 48 h; scoringat 72 hours).
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(173)

Two patients with chronic idiopathic urticaria in whom
remissions were achieved with dye- and preservative-elimination diet had exacerbations of their urticaria when they
were challenged under double-blind, placebo-controlled
conditions with 2,6-di-tert-butyl-p-cresol.
Consuming 2,6-di-tert-butyl-p-cresol free diets, there wasmarked abatement of the frequency, severity and duration of
their urticaria.
Skin irritation and sensitization(4) not assignable
Critical study for SIDS endpoint(174)

UNEP PUBLICATIONS

157

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Remark:SourceReliability
Flag
30.10.2001:::

Remark:Reliability
Flag
30.10.2001::

Remark:An approach was made to estimate the maximal daily intake,assuming that food contained the maximum permitted level of
2,6-di-tert-butyl-p-cresol at the moment of consumption.
Data from a nationwide dietary record survey, carried out in
The Netherlands in 1987/1988, were used. The authors stated,that it cannot be excluded that the acceptable daily intake(ADI) established by the FAO/WHO Joint Expert Committee on
Food Additives (JECFA; 0 - 0.125 mg/kg bw, temporary) andthe Scientific Committee for Food of the Commission of theEuropean Communities (0 - 0.05 mg/kg bw) was surpassed
theoretically in all ages and in both sexes. It should be
noted, that in reality the daily dietary intake would
actually be lower than the ADI because of- the use of no or combinations with other antioxidants inthe surveyed food- losses during food processing (e.g. pastry).
Daily intake
Shell(2) valid with restrictions
Critical study for SIDS endpoint(175) (176) (177) (178)

For a 2-year period (September 1987 to December 1989) 1336
consecutive eczema patients were patch tested. The patches
had been left on the skin for 2 days and readings had been
performed after 2 days, 3 days, and 1 week. All patch tests
were negative. (There was a frequency of 27 % withoccupational or doubtful occupational relevance, which
compares to other studies.)
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(69)

An evaluation of National Intake Assessments of BHT(Information was provided by 10 countries. The submitted
intake assessments were based on ´poundage´, household
economic surveys, sales data, model diets or individual
dietry records.

24.11.2000

Remark

158

(179)

:

Both carcinogenic and anticarcinogenic properties have beenreported for the synthetic antioxidants butylatedhydrocyanisole (BHA) and butylated hydroxytoluene (BHT). The
association between dietary intake of BHA and BHT andstomach caner risk was investigated in the Netherlands
Cohort Study (NLCS) that started in 1986 among 120,852 men
and women aged 55 to 69 years. A semi-quantitative foodfrequency questionary was used to assess food consumption.
Information on BHA or BHT content of cooking fats, oils,
mayonnaise and other creamy salad dressings and dried soups
was obtained by chemical analysis, a Dutch database of foodadditives (ALBA) and the Dutch Compendium of Foods and Diet
Products. After 6.3 years of follow-up, complete data on BHA
and BHT intake of 192 incident stomach cancer cases and 2035
subcohort members were available for case-cohort analysis.
Mean intake of BHA or BHT among subcohort members was 105

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Reliability
Flag
30.10.2001::

Remark:and 351 ug/day, respectively. For consuption of mayonnaise
and other creamy salad dressings with BHA or BHT no
association with stomach cancer risk was observed. A
statistically non-significant decrease in stomach cancer
risk was obsered with increasing BHA and BHT intake [rateratio (RR) highest/lowest intake of BHA = 0.57 (95%
conficence interval (CI): 0.25-1.30] and BHT = 0.74 (95% CI:
0.38-1.43). In this study, no significant associtation withstomach cancer risk was found for usual intake of low levelsof BHA and BHT.
Epidemiology(2) valid with restrictions
Critical study for SIDS endpoint(180)

Based on the results of 126 patients (dental technicians) inthe diagnostic patch test two series for patch testing ofdental technicians are recommended: the "major series" whichcontains acrylates and methacrylates (15 materials) and the"additional series" various additives (among 13 materials
BHT is mentioned). 1 of 103 patients tested (0.9 %) showed apositive reaction to BHT.

30.10.2001(181) (182)Remark:Reliability
Flag
30.10.2001::

Remark:Reliability
Flag
30.10.2001::

Remark:358 patients exposed to plastics and remitted to an
occupational dermatology clinic were patch tested during a
6-year period. Conventional patch-test techniques were used.
No allergic patch-test reactions with BHT were reported.
2/358 showed irritant patch-test reactions.
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(183)

173 patients exposed to plastics and glues were patch tested
during a 3-year period. Conventional patch-test techniques
were used. No allergic patch-test reactions with BHT werereported. 2/173 showed irritant patch-test reactions.
Skin irritation and sensitization(2) valid with restrictions
Critical study for SIDS endpoint(184)

A 37-year old woman had developed an itchy inflammatory
oedema of her scalp and face, with occipital lymph nodeswelling, 1 day after the 2nd application of a new
hair-colouring preparation. Patch testing was performed withthe 20 ingredients of the hair dye. Among others she had apositive reaction at day 2 and day 4 to BHT.

27.11.2000
5.11

(74)

ADDITIONAL REMARKS

Type

:

Biochemical or cellular interactions

UNEP PUBLICATIONS

159

OECD SIDS
5. TOXICITY

Remark2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

Because of the influence of 2,6-di-tert-butyl-p-cresol on
various enzyme systems, and probably caused by its antioxidative properties, 2,6-di-tert-butyl-p-cresol modifies
the action of many other substances on the organism.
A protective effect against many acutely toxic chemicals wasoften observed.

08.09.2001(185) (97)

Type

:

Biochemical or cellular interactions

Remark:Radiosensitizing and radioprotective effects of 2,6-di-tertbutyl-p-cresol have been reported.

21.04.1994

(97)

Type

:

Biochemical or cellular interactions

Remark:The ability of BHT to act either as a suppressor ofstimulated lymphocytes or enhance the inhibition of steroid
immunosuppressors (cortisol and prednisolone) was examined.

27.11.2000

(186)

Type

:

Biochemical or cellular interactions

Remark:The ability of BHT to modulate both phase I and phase II
metabolizing enzymes was examined.

27.11.2000

(187)

Type

:

Biochemical or cellular interactions

Remark:The purpose of the study is to present data which indicatethat selective protein alkylation occurs in rat liver tissue
exposed to BHT under a variety of conditions.

27.11.2000

(188)

Type

:

Biochemical or cellular interactions

Remark:In rats injected with BHT (once or twice 60 mg/kg bw i.p.
injection) the following parameters were determined:
5-methyl-deoxycytidine content in total DNA; AdoMet and
AdoHey contents, the methyl transferase activity, themethylation status and transcription level of the hepatic
methyl transferase gene.

27.11.2000

(189)

Type

:

Biochemical or cellular interactions

Remark:BHT was active in the MCF-7 cell proliferation assay causing
an approximately 2.2-fold increase in cell numbers.

27.11.2000

(190)

Type

:

Biochemical or cellular interactions

Remark:Male Sprague-Dawley rats and male ICR mice were administered
BHT quinone methide orally. BHT quinone methide, is a major
unconjugated metabolite in the liver of rats given BHT. 24
or 48 h later plasma concentrations of blood coagulation
factors were determined.
The substance caused a decrease in several blood clotting

160

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
factors in a dose-dependent manner after 48 h, while asimilar dose of BHT did not.
The findings support the hypothesis that BHT quinone methide
is the active metabolite affecting the vitamin K redox
cycle.

08.09.2001

(191)

Type

:

Chemobiokinetics general studies

Remark:Several in vivo studies verified the importance of themetabolization of 2,6-di-tert-butyl-p-cresol by cytochrome
P450 dependent enzymes for its toxicity and found adetoxifying effect of glutathion and other SH-agents:- Buthionine sulfoxime (BSO), an inhibitor of glutathion
synthesis, enhanced the hepatotoxic effect (increase inserum transaminases) of 2,6-di-tert-butyl-p-cresol inrats and mice, whereas a pretreatment with cysteine
reduced the effect.
- The combined effect of BSO and 2,6-di-tert-butyl-p-cresol
was inhibited in mice by application of drug metabolism
inhibitors such as SKF 525 A, piperonyl butoxide or carbon
disulfide, whereas inductors like cedar wood oil or
phenobarbital tended to increase hepatic injury.
The increased toxicity fo 2,6-di-tert-butyl-p-cresol byphenobarbital was more obvious in male than in female
rats.
- In contrast, injection of cedrene, 6 hours prior to an
i.p. injection of 2,6-di-tert-butyl-p-cresol, prevented
the toxic effect of the test substance on mouse lung.
Cedrene is a sesquiterpene derived from cedar wood, which
stimulates xenobiotic metabolism by inducing microsomal
mixed function oxidases.
(192) (193) (194) (195)

Type

:

Cytotoxicity

Remark:The effect of 2,6-di-tert-butyl-p-cresol (BC) on
differentiation and proliferation in mouse teratoma XB2 cell
cultures in low calcium medium has been assessed by cellcount, relative keratin production and stratification. At aconcentration range of 0.0001 - 10.0 ug/ml BC purely caused
hyperproliferation up to a maximum of approx. 150 % ofcontrol value with no attendant changes in relative keratin
production or stratification.

27.04.1994

22.04.1994

(196)

Type

:

Cytotoxicity

Remark:Cytotoxicity assays were conducted with V79 fibroblasts
exposed to 50 uM 2,6-di-tert-butyl-p-cresol for up to
2 hours:[3H]thymidine uptake and incorporation to DNA:
104 % radioactivity in the nucleotide pool compared tocontrol, 93 % radioactivity in DNA compared to control (meanvalues of three independent trials);[3H]leucine incorporation into proteins:
synthesis was blocked completely at a concentration lower
than 50 uM;
oxygen consumption:
the cells were exposed directly inside the oxygraph chamber,

UNEP PUBLICATIONS

161

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
the effect was recorded immediately during 5 min of
exposure: 2,6-di-tert-butyl-p-cresol treated cells showed
40 % of the control oxygen consumption (recovery to 90 % ofcontrol after 24 hours).

21.04.1994

(197)

Type

:

Cytotoxicity

Remark:Neutral red acute cytotoxicity assays revealed the following
NR50 values(uM concentration of 2,6-di-tert-butyl-p-cresol that
reduced the absorbance (540 nm) of neutral red extracted
from the treated cells by 50 % of control values during a 1or 2-day exposure period):
1 day 2 day
human foreskin fibroblast (HFF) cells
46
34
human melanoma (SK-Mel/27) cells
65
57
normal human epidermal keratinocytes (NHEK) 53
37
normal human epidermal melanocytes (NHEM) 65
45

21.04.1994

(198)

Type

:

Cytotoxicity

Remark:Source28.04.1994:

6-tert-butyl-2-(hydroxy-tert-butyl)-p-cresol (BC-butOH) was
considerably more toxic to bronchiolar Clara cells than
2,6-di-tert-butyl-p-cresol (BC), the compound it was
metabolized from;BC-butOH decreased cell viability to the same extent within
2.5 hours as did a one magnitude higher concentration of BC(the cytotoxic effect was time- and dose-dependent).
Cytotoxicity of BC was found to be depressed by a cytochrome
P450 inhibitor.
These results support the hypothesis that the metabolites ofBC (BC-butOH is readily metabolized to BC-butOH-quinone
methide) are the active compounds that generates acutepneumotoxicity and modulates lung tumour formation.
Shell

Type

:

Cytotoxicity

Remark:The side-chain (at the tert-butyl moiety) hydroxylated
metabolite (BC-butOH) of 2,6-di-tert-butyl-p-cresol (BC) was
found to be ca. 10-fold more cytotoxic in isolated rat
hepatocytes at a 10-fold lower concentration than the parent
compound (BC, e.g. 3 mM).
A similar enhanced cytotoxicity was exhibited by the hydroperoxides of BC and BC-butOH.

(199) (200)27.04.1994(201) (200)

Type

:

Distribution

Remark

:A 50-day administration of 0.5 % 2,6-di-tert-butyl-p-cresol
in diet to rats (ca. 375 mg/kg bw day) led to a test
substance concentration in adipose tissue of 30 - 45 mg/kg
wet weight.

26.03.2003
Type

162

(97)
:

Distribution

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY
Remark2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

Long-term feeding led to accumulation of 2,6-di-tert-butylp-cresol especially in the adipose tissue, and, to a lower
extent, in the liver. The elimination half-time was 7 - 10days in adipose tissue and liver after ceasing of theadministration of test substance.

12.04.1994

(97)

Type

:

Excretion

Remark:2,6-di-tert-butyl-p-cresol was preponderantly excreted withurine and faeces, in a species- and sex-dependent manner.
After a single oral administration to rats 80 - 90 % of theapplied radiolabelled dose was found in urine within 4 days,in rabbits ca. 54 % occurred within 4 days, and in humans
ca. 66 % were found in urine within 11 days (half of thisamount occurred within the first 24 hours).
In rabbits 66 % of the radiolabelled dose was eliminatedwith the urine within 7 days, independently from the routeof administration (dermal, oral, i.v., i.m., dose: 5
mg/animal each route); maximal 20 % appeared in faeces. The
release was incomplete after one week.
7 days after a single i.v. injection of 5 mg test substanceinto guinea pigs, the highest concentration of 14C-labelled
2,6-di-tert-butyl-p-cresol was found in bile (ca. 5 ug/g)
and adipose tissue (ca. 1.3 ug/g).
(202) (97) (98)

Type

:

Immunotoxicity

Remark:Immunosuppressive effects of 2,6-di-tert-butyl-p-cresol were
found in vitro (no further information available).

27.04.1994

12.04.1994

(203)

Type

:

Metabolism

Remark:In rat a marked enterohepatic circulation, especially of the
2,6-di-tert-butyl-p-cresol (BC) metabolite BC-acid and its
glucoronide, was found. An analogous circulation was
suggested in humans.

12.04.1994

(97)

Type

:

Metabolism

Remark:In vitro metabolism of radiolabelled 2,6-di-tert-butyl-pcresol (BC) has been studied with liver and lung microsomesfrom phenobarbital-pretreated rats and mice. 12 metabolites
had been determined qualitatively and quantitatively, whichwere formed by hydroxylation of the alkyl substituents(products of the 4-methyl-group oxidation were BC-alcohol,
BC-aldehyde and BC-acid, the tert-butyl groups were oxidizedin the same manner) and by oxidation of the aromaticring.
Whereas in rat liver and lung oxidation of the ringsubstituted 4-methyl group predominated (BC-4-OH was theprincipal metabolite), in mouse liver oxidation of the
4-methyl- and tert-butyl-groups occurred in a similar
extent. In mouse lung the side-chain (tert-butyl-group)oxidation predominated. Mouse lung, however, produced morequinone derivatives (ring oxidation products) relative toother metabolites than mouse liver or rat liver and lung.

UNEP PUBLICATIONS

163

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
Besides these species-related differences, also micestrain-specific differences in the oxidation reactions were
found: C3H mice showed particularly high oxidation activity(ring, side-chain, 4-methyl-group) in liver; 129/J and A/Jmice showed higher quinone formation activity in liver andlung (factor 2 - 3) as other mouse strains.

21.04.1994

(204)

Type

:

Metabolism

Remark:Source28.04.1994:

The principal metabolites of 2,6-di-tert-butyl-p-cresol (BC)in mouse bronchiolar Clara cells were 6-tert-butyl-2(hydroxy-tert-butyl)p-cresol (BC-butOH; 4.4 +- 1.1 pmol/10E6
cells/minute) and 2,6-di-tert-butyl-p-hydroxymethyl-phenol(BC-OH; 1.0 +- 0.2 pmol/10E6 cells/minute). This metabolite
pattern is nearly identical with that obtained withmicrosomes prepared from whole lungs.
Quinone methide production occurred more readily fromBC-butOH than from BC (0.52 +- 0.14 compared to 0.41 +- 0.06
pmol/10E6 cells/minute). The maximum concentration of theintermediate BC-butOH was very low relative to that of BC;
similar quantities of the quinone methides were generated.
Furthermore, two glutathion conjugates, expected from attackof BC-quinone methide and BC-butOH-quinone methide, were
found.
Incubation time was 15 minutes (Clara cells) or 10 minutes(microsomes).
Shell

Type

:

Metabolism

Remark:2,6-di-tert-butyl-p-cresol (BC) conjugates predominantly
were glucuronides of BC-acid, and, to a lower extent, ofBC-hydroquinones.
Furthermore, a glutathion conjugate of the quinone methide
and the corresponding mercapturic compound was determined.

(199)

12.04.1994

(205)

Type

:

Metabolism

Remark:The pulmonary NAD(P)H-quinone oxidoreductase (QR) activity
was significantly higher in rat than in A/J or C57 mice.
The enzyme can be induced by an i.p. administration of
100 mg/kg bw. High activity of the QR was observed inthree alveolar type II cell lines in vitro.
Maybe the low sensitivity of the rat to 2,6-di-tert-butylp-cresol was due to different QR activities in lungs of miceand rats, because this enzyme is involved in the detoxification of reactive test substance metabolites (e.g.
2,6-di-tert-butyl-quinone methide).

27.04.1994

(206)

Type

:

Toxicokinetics

Remark:2,6-di-tert-butyl-p-cresol (BC) was metabolized to 2,6-ditert-butyl-quinone methide in rat liver. In vitro thephylloquinone (vitamin K1) epoxide reductase, a key enzymeof the vitamin K redox cycle, is inhibited by the BC-quinone
methide, but not by BC itself. The quinone methide also

164

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
inhibits the phylloquinone epoxide-dependent protein
carboxylation, so that the post-translational protein modification of blood coagulation factors II (prothrombin), VII,
IX and X is inhibited.
The hepatic BC-quinone methide concentration correlates withthe plasma concentration of these coagulation factors andlikewise with the species and sex differences in the BC
effect.
Livers of BC-resistent ICR mice may have very little ability
to oxidize BC to its corresponding quinone methide. However,administration of BC-quinone methide to mice of this strain
also leads to an inhibition of the blood clotting.

21.04.1994(207) (208)

Type

:

Toxicokinetics

Remark:2,6-di-tert-butyl-p-cresol causes necrosis in mouse alveolar
type I cells followed by an increased formation of alveolar
type II cells during a repair period.
Prior to the lung injury a covalent binding of 2,6-di-tertbutyl-p-cresol or one of its metabolites (maybe quinon
methide) to the lung tissue occurred, so that a causal
relationship between the macromolecular binding and the lunginjury has been suggested.
(97) (209) (210)

Type

:

Toxicokinetics

Remark:The lethal effect of 2,6-di-tert-butyl-p-cresol at higher
doses is caused by lung injuries and haemorrhage due to theinhibition of blood clotting. It was suggested that thehaemorrhagic diathesis is basing on a vitamin K-antagonism,
possibly caused by the 2,6-di-tert-butyl-p-cresol metabolite
2,6-di-tert-butyl-quinon methide. The vitamin K-dependent
blood coagulation factors II (prothrombin), VII, IX and X
decreased. Vitamin K shows a protective effect.

21.04.1994

21.04.1994

(97)

Type

:

other

Remark:2,6-di-tert-butyl-p-cresol showed anti-virus action andprolonged the lifespan of Drosophila, rat and mouse.

27.04.1994

(97)

Type

:

other

Remark

:A summary of the numerous studies on mutagenicity/
genotoxicity

15.11.2000

(96)

Type

:

other

Remark27.11.2000:

revision: 11/00

Type

:

other

Remark:Reliability:

An overall perspective of the toxicology of BHT is given inthese comprehensive publications.
(4) not assignable

UNEP PUBLICATIONS

165

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Flag
30.10.2001

:

Type

:

other

Remark:In several publications BHT is used as an well known exampleof phenolic substrate or antioxidants.
(213) (214) (215) (216) (217) (118) (218) (219) (220) (221) (222) (223)

Type

:

other: Effect of antioxidant on liver cell maturation

Remark:Male rats (F344) of different ages were fed diet containing
0.5 % BHT. Effects on body weight, nuclear ploidy and GGTacticity were determined. BHT caused a marked reduction inbody weight gain, it did not affect ploidy as expected, it
induced GGT rapidly.

Critical study for SIDS endpoint(211) (11) (212)31.01.2001

28.05.1994

(224)

Type

:

other: Effects on biochemical parameters

Remark:BHT accelerates retinoic acid-induced maturation of the
HL-60 human leukemia in vitro.

09.11.1998

(225)

Type

:

other: Effects on biochemical parameters

Remark:The publication describes the effects of short term exposuteto tert.-butyl hydroperoxide upon human erythrocyte Rb+
fluxes and internal sodium and potassium concentrations andthe protective action of BHT among each other.

09.11.1998

(226)

Type

:

other: Effects on biochemical parameters

Remark:BHT had no significant effect on collagen metabolism andcell growth in normal fibroblasts.

09.11.1998

(227)

Type

:

other: Effects on biochemical parameters

Remark:The addition of BHT to isolated rat hepatocytes caused aconcentration (0.5, 1.0 mM)-dependent acute cell death. The
toxicities were accompanied by the loss of cellular ATP and
GSH.

09.11.1998

(228)

Type

:

other: Effects on enzymes

Remark:The study investigated the effects of BHT (0.05% and 0.15 %in the diet for 14 days) on the induction of hepatic
drug-metabolizing enzymes in mice and hamsters. BHT has apotency to induce specific isoenzymes of mono-oxygenases andtransferases. The induction mode of the isoenzymes was notsimilar between the mouse and Chinese hamster.

09.11.1998

(229)

Type

:

other: Effects on enzymes

Remark:The study demonstrated that co-administration of BHT (0.1%166

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
in the diet for 14 days) elevated significantly theactivation of aflatoxin B1 and Benz[a]pyrene in the mouse
liver (determined by the mutagenicity test)

09.11.1998

(230)

Type

:

other: Excretion and distribution

Remark:Single oral doses of 20, 63 or 200 mg 2,6-di-tert-butyl-pcresol (BC)/kg bw were administered to rats.
Maximum plasma concentrations of test substance wereobserved after 2 - 4 hours in the two highest dose groups(in the low dose group the level was around the limit ofdetection with no clear peak). The area under the plasma
concentration-time curve (AVC; 0 - 8 hours after administration) was increased in a dose-dependent manner.
1.0 +- 0.4 % of the applied dose were excreted within days
1 - 4 as unconjugated BC acid in the urine,
1.1 +- 0.4 % as (un)conjugated BC acid in the urine, and9.9 +- 9.1 % as unchanged test substance in the faeces.

28.04.1994

(165)

Type

:

other: Gap junctional intercellular communication test

Remark:Non-transformed (C10) mouse lung epithelial cells and SV-40transformed Djungarian hamster DM15 fibroblasts,
respectively were treated with 2,6-di-tert-butyl-p-cresol(BC) for 4 or 14 hours. Then, Lucifer Yellow fluorescent dye
was microinjected into cells and the number of directly
adjacent cells to which dye had spread was quantified. At 33- 55 ug/ml intercellular communication was significantly
inhibited compared to control cultures in mouse lung cells.
In hamster fibroblasts BC was an effective uncoupler atconcentrations as low as 10 ug/ml.
The optimal effect was observed at 50 - 100 ug/ml.
It was suggested, that this effect may be important in themechanism of tumour promotion.

27.04.1994(231) (232)

Type

:

other: Gap junctional intercellular communication test

Remark

:6-Thioguanine (TG) resistant adult rat liver (ARL) cells
were co-cultured with 6-TG sensitive hepatocytes in presenceof 6-TG. Addition of test substance led to a dose-related
increase in cell survival after a 2-day incubation comparedto the negative control (87 % compared to 64 % survival).
Survival was similar to that in the positive control (0.35
ug DDT/ml; 83 % survival), so that 2,6-di-tert-butyl-pcresol was clearly positive in this cell communication test.
It is suggested, that this effect may be important in themechanism of tumour promotion.

21.04.1994

(91)

Type

:

other: Immunotoxicity

Remark:This study indicated that induction of enzyme-alteredpreneoplastic liver foci (EAF) in F344 rats resulted in astatistically significant reduction in the natural killer(NK) cell activity of spleen mononuclear cells tested at 5wk
following EAF induction compared with saline controls. The
addition of 0.5% BHT to the diet also resulted in a

UNEP PUBLICATIONS

167

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
significant decrease in the splenic NK cell activity
compared with the rats fed the control diet
Summary: the activity of NK cells was definitely suppressed
during (a) induction of EAF and (b) promotion of EAF by BHT
treatment

24.11.2000

(233)

Type

:

other: In vitro DNA synthesis inhibition test

Remark:The concentration of test substance which inhibits DNA
synthesis by 50 % in HeLa S3 cells was 500 uM (measured as
decrease in BrdUrd-labelling); 90-min incubation.

20.04.1994

(234)

Type

:

other: In vitro DNA-binding inhibition test

Remark:Incubation of rat liver microsomes with up to ca. 12 mM
2,6-di-tert-butyl-p-cresol and [3H]2-acetylaminofluorene(2-AAF) or [3H]N-OH-2-acetylaminofluorene (N-OH-2-AFF) for
60 minutes led to a decrease in binding of these radiolabelled tumour inductors to calf thymus DNA compared tocontrol cultures without 2,6-di-tert-butyl-p-cresol. In rat
primary hepatocytes incubated with 2-AAF, the same effectcould be achieved, being significant in all treated assays(0.01, 0.025, 0.05 and 0.1 uM 2,6-di-tert-butyl-p-cresol;
20-hour incubation).

12.04.1994

(235)

Type

:

other: In vitro effects on DNA-binding

Remark:Test substance
21.04.1994:

NADPH-dependent binding of 3,3'-dichlorobenzidine (DCB) to
calf-thymus DNA in a S9-activated test system was enhancedby 2,6-di-tert-butyl-p-cresol; at 4, 20 and 100 uM test
substance, the enhancement of DCB-DNA-binding was 76, 328
and 328 %, respectively.
vehicle: methanol

Type

:

other: In vivo DNA-binding inhibition test

Remark:Source28.04.1994:

Dietary 2,6-di-tert-butyl-p-cresol (BC) can inhibit 7,12dimethylbenz[a]anthracene (DMBA)-DNA binding in mammary
tissue of rats, measured at 18 or 22 hours following DMBA
administration. Female rats had been fed a diet containing
0.4, 0.6 or 0.8 % test substance in diet 25 or 24 days prior
to gastric intubation of 55.5 mg [3H]DMBA or 31.6 mg DMBA/kg
bw (DMBA is an inductor of mammary carcinogenesis).
The controls were fed an unsupplemented diet prior to DMBA
intubation.
Shell

Type

:

other: In vivo effects on DNA synthesis

Remark:2,6-di-tert-butyl-p-cresol did not elicit a significant
increase or decrease in DNA synthesis (BrdUrd-labelling
index: 0.320 +- 0.415 % compared to the control index of
0.120 +- 0.179 %) in renal pelvic epithelium of rats. The
rats had been fed with 1 % test substance in diet for 4
weeks before they were injected i.p. with BrdUrd and

168

(236)

(237) (238)

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
subsequently killed by exsanguination; 1000 cells were
evaluated; 10 animals/dose and control group.

25.04.1994

(239)

Type

:

other: In vivo nucleic acid-binding test

Remark

:8 hours after a single oral administration of 55 mg
2,6-di-([U-14C]tert-butyl)-p-cresol/kg bw (20 uCi/animal) to
mice a covalent binding of the labelled substance to liver
RNA was found. Adducts of the purine nucleotides AMP and GMP
were the major labelled products, but ca. 15 % of theradioactivity was not bound to any nucleotide.

20.04.1994

(240)

Type

:

other: Mechanism of tumour promotion

Remark:The nature of the skin tumour promotion response to a hydroperoxide metabolite of 2,6-di-tert-butyl-p-cresol,
2,6-di-tert-butyl-4-hydroperoxyl-2,5-cyclohexadienone(BCOOH), was examined in SENCAR mice. BCOOH was aneffective inducer of epidermal ornithine decarboxylase (ODC)
activity after a single application. Dose-dependent
increases were seen between 2 and 20 umol while higher
amounts were less effective. A similar dose-response
relationship for papilloma and carcinoma formation wasobserved when BCOOH was applied twice weekly for 50 weeks
to mice previously initiated with 7,12-dimethylbenz(alpha)anthracene. However, BCOOH is not a complete carcinogen inthat no papillomas or carcinomas were observed inuninitiated mice treated with BCOOH only.

22.04.1994

(241)

Type

:

other: Mechanism of tumour promotion

Remark:An investigation of changes in urine composition, morphologyof bladder epithelium and levels of DNA synthesis caused by
2,6-di-tert-butyl-p-cresol (BC) was performed on maleFischer 344 rats. 10 animals/dose and control group received
BC, respectively at a concentration level of 1 % in the dietor basal diet without any chemical supplement. Five rats ineach group were killed after 4 weeks for estimation of DNA
synthesis level and histologic examination by lightmicroscopy and at 8 weeks for morphologic examination by
light microscopy and scanning electron microscopy (SEM) inthe urinary bladder. A significant decrease in body weight
was observed for the BC treated group at weeks 4 and 8 ascompared to the control group values. Rats given BC for 4
weeks had increased phosphorus and MgNH4PO4 crystals in theurine. Urinary bladder DNA synthesis was significantly
increased. Macroscopic abnormalities of the urinary bladderwere observed neither at week 4 nor at week 8 and no histopathological lesions were evident at week 4. Leafy or ropy
microridges and/or short uniform microvilli were observed by
SEM on the bladder epithelial surface of rats given BC.
These results probably provide a link among promotion of
bladder carcinogenesis, increased DNA synthesis and alteredsurface morphology after administration of BC.

28.04.1994
Type

(242)
:

other: Mechanism of tumour promotion

UNEP PUBLICATIONS

169

OECD SIDS
5. TOXICITY

Remark2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

The potency of a hydroxyperoxide metabolite of 2,6-di-tertbutyl-p-cresol, 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl2,5-cyclohexadienone (BCOOH), as an inducer of ornithine
decarboxylase (ODC) activity and as skin tumour promoter wasinvestigated in female SENCAR mice:
BCOOH caused a dose-dependent induction of epidermal ODC
activity following application of 1, 4, 8, 20 or 40 umol tothe shaved dorsal skin of 8 animals per treatment group.
In a two-stage tumour promotion study the animals were
initiated on the shaved dorsal skin with 20 nmol of 7,12-dimethylbenz(alpha)anthracene. The first stage promotion had
begun 10 days later and consisted of twice weekly treatmentsof 2 ug phorbol-12-myristate 13-acetate for two weeks.
Thereafter, the mice were exposed to BCOOH twice weekly by
application to the dorsal skin at dosage levels of 8 or
20 umol. This second-stage promotion treatment was carriedout with 25 animals per dose. Treatment continued for a
total of 24 weeks of first- and second-stage promotion.
Following this treatment regimen BCOOH caused a 64 %incidence of papillomas with an average tumour burden of 1.9
tumours per mouse at termination.

28.04.1994

(243)

Type

:

other: Mechanism of tumour promotion

Remark:Microsomal metabolites of 2,6-di-tert-butyl-p-cresol (BC) in
four different mice strains were analyzed and the relative
amounts of BC-butOH (hydroxylated at one tert-butyl group),
BC-OH (hydroxylated at the p-methyl group) and 2,6-di-tertbutyl-1,4-benzoquinone were determined.
An excellent correlation was obtained (r = 0.999) between
the amounts of BC-butOH produced and the susceptibility ofthese strains to BC tumour promotion as measured by thepercentage change in tumour number after induction withurethane. No correlation existed between tumour promotion
and formation of the other two metabolites.
Direct evidence for the role of BC-butOH in tumour promotionin Ma/MyJ mice was given by i.p. administration of thismetabolite. 50 mg/kg bw enhanced the urethane-initiated lungtumour formation to about the same degree as 4-fold larger
doses of 2,6-di-tert-butyl-p-cresol.
The treatment regimen for all mice involved a single i.p.
injection of urethane followed by 6 weekly injections of
the potential promotor (50 mg BC-butOH/kg bw, all others:
200 mg/kg bw).

27.04.1994

(244)

Type

:

other: Metabolism and biochemical interactions

Remark:Radiolabelled 2,6-di-tert-butyl-p-cresol was metabolized to
2,6-di-tert-butyl-quinone methide by a peroxidase in vitro.
The metabolite dimerized to stilbenquinone or boundcovalently to microsomal protein. Phenolic compounds in thein vitro assay enhanced this effect.
It was suggested that a radical precursor of the quinon
methide might be responsible for covalent binding in absenceof any activators. In the presence of activators, theincrease in the formation of the quinon methide may accountfor the increase in covalent binding. Thus, both a free170

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
radical of 2,6-di-tert-butyl-p-cresol and the quinon methide
may be involved in the covalent binding to tissue macromolecules and hence its toxicity.

27.04.1994(209) (210)

Type

:

other: Resorption

Remark:2,6-di-tert-butyl-p-cresol is easily resorbable from theintestines, and can be resorbed via the skin in small
amounts.

12.04.1994

(97)

Type

:

other: Resorption

Remark:2,6-di-tert-butyl-p-cresol crosses the placental barrier andis found to be excreted in rat milk.

21.04.1994(98) (79)Type:other: Review

Remark:Reliability
Flag
24.07.2001::

Butylated hydroxytouene (BHT) is widley used antioxidant
food additives. It has been extensively studied forpotential toxicities. This rewiew details experimental
studies of genotoxicity and carcinogenicity which bear on
cancer hazard assessment of exposure to humans. We conclude
that BHT pose no cancer hazard and, to the contrary, maybe
anticarcinogenic at current levels of food additive use.
(4) not assignable
Critical study for SIDS endpoint

Type

:

other: Review

Remark:Although in vitro data have previously been consideredduring the risk assessment of food additives, they havegenerally had no direct influence on the calculation of ADI
values.In this review 18 food additives (among others BHT)
are evaluated for the availabity of in vitro toxicity data
which might be used for the derivation of a specificdata-derived uncertainty factor(245)

24.11.2000

(246)

Type

:

other: Sensitization

Remark:

BHT, which is an antioxidant additive in the major jet fuels
used, reduced the skin sensitization potential of 1 of two
fuels examined in the murine Local lymph node assay (LLNA)

24.11.2000

(247)

Type

:

other: Special studies on hepatotoxicity

Remark:It was demonstrated that feeding 0.5% dietary BHT for 30days after the induction enzyme-altered foci led to a 20- to
30-fold increase in the
gamma-glutamyltranspeptidase-positive areas in bothdiethylnitrosamine and saline-initiated rat livers, but nomajor effects in glutathione S-transferase placental form
positive foci.

09.11.1998(248)

UNEP PUBLICATIONS

171

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type

:

other: Special studies on potentiation of cancer

Remark:Chronic BHT treatment after a single administration of a
carcinogen increases lung tumor multiplicity in some inbred
strains of mice.

09.11.1998

(249)

Type

:

other: Special studies on potentiation of cancer

Remark:The study demonstrated the initiation and promotion process
by using 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone in
the drinking water as the initiator and BHT in the diet as
the promoter in the development of lung tumors in the A/Jmouse.

09.11.1998

(250)

Type

:

other: Special studies on potentiation of cancer

Remark:Mice given 10 ug/g bw of 3-methylcholanthrene (MCA) develop
no lung tumors unless this is followed by chronic BHT
exposure (six weekly i.p. injections of 200 mg/kg bw. Slightly higher MCA
doses induce low lung tumor multiplicities that are increased 12-26-fold bychronic BHT administration.
(251)

Type

:

other: Special studies on pulmonary toxicity

Remark:Examination of lung morphology and biochemistry after acuteand multiple i.p injections in 2 inbred mouse strains.

26.03.2003

09.11.1998

(252)

Type

:

other: Special studies on pulmonary toxicity

Remark:

The time course of changes in the pulmonary contents ofprotein kinase C and the calcium-dependent protease ,calpain, after i.p. injection (200 mg/kg bw) in mice wasstudied in detail to explore the potential of using these
enzymes as biomarkers for lung injury.

26.03.2003

(252)

Type

:

other: Special study

Remark:BHT inhibited gap junctional intercellular communication in
WB-F344 rat liver and C10 mouse lung epithelial cells in adose and time-dependent manner.

09.11.1998

(253)

Type

:

other: Special study

Remark:The effect of BHT on the stimulation of lymphocytes in thepresence of cortisol and prednisolone was determined.
Concentrations of BHT over 100 ul/mL were shown to have a
toxic effect on lymphocytes. A dose related synergistic
effect was observed with cortisol or prednisolone in
combination with BHT.

09.11.1998
Type

172

(186)
:

other: Special study on haemorrghagic effects

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY
Remark2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004:

The study demonstrated that a high dose of BHT (3 g/kg bw day for up to
14 or 21 days) in the diet of rats causes modest haematological effects inaddition to the reported reduction of vitamin K-dependent clotting factors.
(254)

Type

:

other: Special study on haemorrghagic effects

Remark

:10-5M and 10-7M BHT decreased the amount of TXB2 synthesisin A-23187 and thrombin-stimulated rabbit washed platelets,
respectively

26.03.2003

09.11.1998

(255)

Type

:

other: Toxicological symptoms

Remark:2,6-di-tert-butyl-p-cresol caused thyroid gland enlargement
and histological changes (microfolliculation), accompanied
by an increased iodine uptake (no further data available).

21.04.1994

(97)

Type

:

other: Tumor inhibition

Remark:Result:

Method: male 6-wk-old F344 rats were given a single i.p.
injection of 200 mg/kg bw of diethylnitrosamine; starting 2
wk later., animals were placed on powdered diet containing
0.03% 2-amino-3.8-dimethylimidazol[4,5-f]quinoxaline (MeIQx)
alone, MeIQx plus BHT at doses of 0.25% each or basal dietalone for 6wk. At wk3 all rats were subjected to 2/3 partial
hepatectomy.; all rats were killed at wk 8
BHT inhibited development of preneoplastic glutathione
S-transferase placental form (GST-P) positive foci ascompared with MeIQx alone.

24.11.2000

(256)

Type

:

other: Tumor promotion

Remark:Result:

Method: BALB/c mice were treated on day 17 of gestation with 5, 15 or 45
mg/kg bw 3-methylcholanthrene and 6 weeks after birth these mice were
given 6 consecutive weekly i.p.
injections of 200 mg/kg bw BHT dissolved in corn oil.. At 6
months of age the mice were killed.
BHT had no statistically significant effects on either tumor
incidence, tumor multiplicity or the mutational spectrum
produced in the Ki-ras gene by in utero MC treatment.
However, though not significant, there was an observable
trend in increased tumor multiplicity in mice co-treatedwith BHT. Unlike what occurs when adult BALB/c mice aretreated with MC, BHT does not appear to significantly
promote the formation of lung tumors following
transplacental exposure to MC, possibly due to the rapid
growth and cell proliferation in the developing organism.
(257)

26.03.2003
Type:other: Tumor promotion

Remark:The abilities of BHT and BHT metabolites to kill
non-tumorigenic and tumorigenic mouse and human lung cellline were compared and the contribution of apoptosis to thiscytotoxicity was examined.

27.11.2000(258)

UNEP PUBLICATIONS173

OECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004

Type

:

other: development of rapid in vivo assay for lung carcinogens

Remark:the goal of the study was to demonstrate the susceptibilityof rasH2 mice to lung urethane carcinogenesis; BHT was used
as tumor promoter

24.11.2000

(259)

Type

:

other: in vitro DNA synthesis inhibition test

Remark:

The test is based on primary cultures of hepatocytes. The
cells have been isolated from enzyme-altered fociEAF)-bearing rats. BHT gave a weak and varaible differential
effect.

27.11.2000

(260)

Type

:

other: in vitro effect on DNA cleavage

Remark:Source28.04.1994:

The effect of 2,6-di-tert-butyl-p-cresol (BC) and its
metabolites on DNA cleavage in vitro was studied withsupercoiled plasmid DNA, pUC18, by agarose gelelectrophoresis. Among several BC metabolites, BC-quinone(2,6-di-tert-butyl-p-benzoquinone) caused cleavage ofsupercoiled DNA (form I) at a concentration as low as 1 uM.
The relative amount of linear form (form III) was increased
with increasing BC-quinone concentration. No DNA cleavage
was detected by BC.
Shell

Type

:

other: in vivo binding effect on biomacromolecules

Remark:Source28.04.1994:

After topical application of an ethanolic solution of 2,6di-tert-butyl-p-cresol (BC) onto the shaved skin of Wistar
rats, labelled 8-MOP (8-methoxypsoralen) was applied using
the same solvent. After this the rats were exposed to UV-A
light. By separating epidermal lipids, DNA/RNA and proteinsby a selective extraction method, irreversible binding of
8-MOP to each of these biomacromolecules was determined. BC
caused a decrease in the photobinding of 8-MOP to epidermal
DNA and proteins.
Shell

Type

:

other: inibition of androgen receptor activation

RemarkResult::

Method: Androgen receptor assay
BHT partially inhibited activation by Salpha-Dihydrotestosterone but alone had no effect.

(261)

(262)

24.11.2000

(263)

Type

:

other: see remark

Remark:To evaluate ability of the precision-cut lung slice model
system to detect species-dependent pneumotoxicity BHT andparaquat were studied as model pneumotoxicants.

27.11.2000
Type
Remark

174

(264)
:
:

other: see remark
The study was designed to establish the extent to which
isoforms CYP2A5, 2E1, "F2 or orthologue(s) of 2B1 are

UNEP PUBLICATIONSOECD SIDS
5. TOXICITY

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004
involved in the pulmonary activation of BHT. Selective
induction and inhibition experiments have been used to
identify the cytochrome P450 isoforms responsible for BHT
bioactivation in mouse lung.

27.11.2000
Type
Remark(189)::

other: sensitization
Guinea pigs sensitized to 2,6-dimethylol p-tert-butylphenol(2,6-MPTBP) showed no cross reaction to BHT.

27.11.2000(265)

UNEP PUBLICATIONS

175

OECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(1)

Chang, S.-S. & Maurey, J.R., J. Chem. Eng. Data 30, 384-387 (1985)(2)

American Conference of Governmental Industrial Hygienists Inc., Cincinnati, Ohio:
Documentation of the threshold limit values and biological exposure indices: 5th ed. (1986),
p. 227(3)

Pardee, W.A. et al., Industrial and Engineering Chemistry36(7), 595 - 603 (1944)(4)

Römpp Lexikon Chemie - Version 2.0, Stuttgart/New York: Georg Thieme Verlag 1999

(5)

The Merck Index, Whitehouse Station, NJ, USA: Windholz M. Merck and Co., Inc.(ed)(1983) 215-216, No. 1521: Butylated Hydroxytoluene(6)Grossmann, N. et al., Journal f. praktische Chemie 330 (2), 204-212 (1988)(7)

Ullmann's Encyclopedia of Industrial Chemistry. Release 2001 6th Edition(8)

Auer Technikum/Auergesellschaft, Berlin, Ausg. 12 (1988), No 1001

(9)

Bayer AG 1973, Internal Study, Test on density, AP-Nr. 514 497 (1973-03-09)(10)

Bayer AG 1986a, Internal Study, Test on vapour pressure, AP-No 682099(11)

BUA Report 58, VCH, February 1991

(12)

Shell 1983, Internal Study, Determination of Partition Coefficient(13)

Bayer AG 2001e, Calculation with SRC-KowWIN v1.66 (2000)(14)

Freese, E. et al., Teratology 20, 413 (1979)(15)

US EPA, Chemical hazard information profile BHT, draft report (1984)(16)

Bayer AG 1986b, Internal Study, Test on water solubility (1986-11-12)(17)Inui, H. et al., Chemosphere 6, 383-391 (1979)(18)

Yoshida, K. et al., Non-steady state equilbrium model for the preliminary prediction of thefate of chemicals in the environment. Ecotox. Environm. Safety 7, 179-190 (1983)(19)

Bayer AG 2001f, Internal Study, Determination of autoflammability of BHT (2001-02-07)(20)

Bayer AG, Internal Study, Report 'Brand- und Explosionsgefahr bei Stäuben' (1987-04-23)(21)

Bayer AG 2001d, Calculation of the Photodegradation of BHT according to SRC-AOPWIN,
v1.90 (2000)(22)

Mikami, N. et al., Chemosphere 5, 311-315 (1979)(23)

Inui, H. et al., Chemosphere 6, 393-404 (1979)(24)

Mikami, N. et al., Chemosphere 5, 305-310 (1979)(25)

Landesanstalt für Umweltschutz Baden-Württemberg, Personal Communication to BUA(1994)(26)

Landesumweltamt Nordrhein-Westfalen, Communication to BUA (1996)

176

UNEP PUBLICATIONSOECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(27)

Bayer AG 2001a, Calculation of the Henry's law constant of BHT according to SRCHENRYWIN, v3.10 (2000)(28)

Jungclaus, G.A. et al., Environ. Sci. Technol. 12, 88-96 (1978)(29)

Bayer AG 2001b, Calculation of the environmental distribution of BHT according to Mackay
fugacity model level I. Method published in: Mackay, D., Multimedia Environmental
Models: The Fugacity Approach. Lewis Publ. Inc., Michigan, U.S.A. (1991)(30)

Bayer AG 2001c, Calculation of the environmental distribution of BHT according to Mackay
fugacity model level I. Method published in: Mackay, D., Multimedia Environmental
Models: The Fugacity Approach. Lewis Publ. Inc., Michigan, U.S.A. (1991)(31)

MITI, Biodegradation and Bioaccumulation Data of Existing Chemicals Based on the CSCL
Japan, Compiled under the Supervision of Chemical Products Safety Division, Basic
Industries Bureau MITI, Ed. by CITI, October 1992. Published by Japan Chemical Industry
Ecology-Toxicology & Information Center(32)

Bayer AG 1975-1980, Internal Study, Prüfung auf biologischen Abbau - Document notavailable(33)

Bayer AG 1984, Internal Study, Bestimmung der letalen Wirkung beim Zebrabärbling
Brachydanio rerio, Verfahrensvorschlag des UBA, Stand 1.6.1982(34)

Bayer AG 1994, Internal Study, Test on acute toxicity to fish, test report 466 A/94

(35)

Shell Research Limited 1982, Butylated hydroxy toluene: Acute toxicity to Salmo gairdneri,
Daphnia magna, and Selenastrum capricornutum; Document Number: SBGR.82.157(36)

Tsuji, S. et al., Eisei Kagaku 32, 46-53 (1986)(37)

Bayer AG 1976, Internal Study, Range finding test with Leuciscus idus

(38)

Verschueren, K., Handbook of environmental data on organic chemicals, 2nd Ed., Van
Nostrand Reinhold Company, p. 467 (1983)(39)

Bayer AG 1994, Internal Study, Acute toxicity of BHT to Daphnia magna, test report 466
A/94

(40)

Passino, D.R.M. & Smith,S.B., Acute Bioassays and hazard evaluation of represantative
contaminants detected in Great Lakes fish. Env. Tox. Chem. 6, 901-907 (1987)(41)

Bayer A 1994, Acute toxicity of BHT to the alga Scenedesmus subspicatus, test report 466
A/94

(42)

Bayer AG 2000, Internal Study, Toxicity of BHT to activated sludge(43)

Bayer AG 1984, Internal Study, Toxicity of BHT to bacteria (Pseudomonas putida)(44)

Trevors, J.T. et al., Bull. Environm. Contam. Toxicol. 27, 433-439 (1981)(45)

Yoshioka, Y. et al., Testing for the Toxicity of Chemicals with Tetrahymena pyriformis, The
Science of the Total Environment, 43, 149-157 (1985)(46)

Ayaz, M. et al., J. Food Protection 43, 4-6 (1980)(47)

Bayer AG 1994, Internal Study, Chronic toxicity of BHT to daphnia magna; test report 466
A/94

UNEP PUBLICATIONS

177OECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(48)

Bayer AG 1986, Internal Study, Chronic toxicity of 2,6-Bis(1,1-dimethylethyl)-4methylphenol in the daphnia reproduction test. Test report from 05.02.1986(49)

Grochowska, M.J. and Buta, G.J., Scientia Horticulturae, 26, 217-224 (1985)(50)

Larson C, Driscoll C, Gross WB (1985) Aflatoxin-Antioxidant Effects on Growth of Young
Chicks. Poultry Science 64, 2287 - 2291

(51)

Osamu Takahashi and Kogo Hiraga, Short-term toxicity of Butylated Hydroxytoluene toJapanese quail, Ann. Rep. Tokyo Metr. Res. Lab. P.H., 32-2, 65-66 (1981)(52)

Hazleton France (1988): Rapport No. 801300 to Rhone-Poulenc S.A.

(53)

Bomhard, E. (1996): J. Am. Coll. Toxicol. 15, S72(54)

Spanjers, M.T., Til, H.P. (1978): Determination of the acute oral toxicity of Vulkanox KB in
rats. Unpublished report to Bayer AG, January 27, 1978

(55)

Deichmann, W.B. et al. (1955): Arch. Ind. Health 11, 93 - 101(56)

Karplyuk, I.A. (1959): Vopr. Pitaniya 18, 24 - 29

(57)

Ichikawa, H. et al. (1976): Ann. Rep. Tokyo Metr. Res. Lab. Public Hlth. 27, 93 - 99

(58)

Baer, V. et al. (1977): Neoplasma 24, 253 - 258(59)Life Sciences Research Office, Federation of American Societies of Experimental Biology,
Bethesda, Maryland (USA) (1973): NTIS/PB 259 917(60)

Bikbulatov, N.T. (1976): Gig. Tr. Zabol. Neft Neftekhim Prom-st, 118 – 121 cited in: Shell
International (1988): Review of toxicology Ionol CP (butylated hydroxytoluene, BHT)

(61)

McOmie, W.A. et al. (1949): J. Am. Pharm. Assoc. Sci. Ed. 38, 366 - 369(62)Hazleton France (1988): Rapport No. 801301 to Rhone-Poulenc S.A.

(63)

Kawano, S. et al. (1981): Toxicol. Appl. Pharmacol. 61, 475 - 479(64)

Kehrer, J.P., DiGiovanni, J. (1990): Toxicol. Lett. 52, 55 - 61

(65)

Yamamoto, K. et al. (1980): Toxicol. Lett. 6, 173 - 175(66)

Witschi, H., Saheb, W. (1974): Proc. Soc. Exp. Biol. Med. 147, 690 - 693(67)

Mallette, F.S., von Haam, E. (1952): A.M.A. Arch. Ind. Hyg. Occup. Med. 5, 311 - 317(68)

Van Beek, L. (1976): CIVO-TNO Report No. R 5041, Working Group on the Toxicity of
Rubber Auxiliaries(69)

Flyvholm, M.-A., Menne, T. (1990): Contact Dermatitis 23, 341 - 345(70)Goh, C.L., Ho, S.F. (1993): Contact Dermatitis 28, 134 - 138(71)

Schnuch, A. et al., British Journal of Dermatology 138, 467-476 (1998)(72)

Motolese, A. & Seidenari S., Contact Dermatitis 30, 49-50 (1994)(73)

Kaniwa, M.-A. et al., Contact Dermatitis 30, 26-34 (1994)(74)

Le Coz C.J. & Schneider, G.A., Cont. Derm. 39, 39-40 (1998)

178

UNEP PUBLICATIONSOECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(75)

Koch, P., Occup. Environ. 44, 62-67 (1996)(76)

Takahashi, O., Hiraga, K. (1978): Toxicol. Appl. Pharmacol. 43, 399 - 406(77)

Takahashi, O., Hiraga, K. (1978): Food Cosmet. Toxicol. 16, 475 - 477(78)

Williams, G.M. et al. (1990): Food Chem. Toxicol. 28, 799 - 806(79)

Olsen, P. et al. (1986): Food Chem. Toxicol. 24, 1 - 12

(80)

Takahashi, O. (1986) Arch. Toxicol. 58, 177-181(81)

Takahashi, O., Hiraga, K. (1979) J. Nutr. 109, 453-457(82)

Powell, C.J. et al. (1986): Food Chem. Toxicol. 24, 1131 - 1143

(83)

Miyakawa, Y. et al. (1986): Toxicol. Lett. 34, 99 - 105(84)

Sondergaard, D. & Olsen, P., Toxicology Letters 10, 239-244 (1982)(85)

McFarlane, M. et al. (1997): Food Chem. Toxicol. 35, 753-767(86)

Price, S.C.(1994); Robens Institute; Report No. RI93/TOX/0020, 29 July 1994

(87)

Inai, K. et al. (1988): Jpn. J. Cancer Res. (Gann) 79, 49 - 58

(88)

Takahashi, O. (1992): Food Chem. Toxicol. 30, 89 - 97

(89)

Allen, J.R. & Engblom, J.F. (1972): Food Cosmet. Toxicol. 20, 769-779(90)

Takahashi, O. et al., Food Cosmet. Toxicol. 18, 229-235 (1980)(91)

Williams, G.M. et al. (1990): Food Chem. Toxicol. 28, 793 - 798(92)

Watanabe, K. et al., Mutat.Res. 416, 169-181 (1998)(93)

Galloway, S.M. et al. (1987): Environ. Mol. Mutagen. 10, Suppl. 10, 1 - 175(94)

Grillo, C.A. & Dulout, F.N. (1995): Mutation Research 345, 73-78(95)

Heil, J. et al., Mutation Research 368, 181-194 (1996)(96)

Bomhard, E.M. et al. (1992): Mutat. Res. 277, 187-200(97)

Deutsche Forschungsgemeinschaft (1985): Toxikologisch- arbeitsmedizinische
Begruendung von MAK-Werten - Butyl- hydroxytoluol (BHT), VCH Weinheim(98)

IARC (1986): Monograph on the evaluation of the carcinogenic
risk of chemicals to humans 40, 161 - 206(99)

Kinae, N. et al. (1981): Water Res. 15, 17 - 24

(100)

Litton Bionetics Inc. (1975): NTIS/PB 245487(101)

Yoshida, Y. (1990): Mutat. Res. 242, 209 - 217(102)

Hageman, G.J. et al. (1988): Mutat. Res. 208, 207 - 211(103)

Hirano, K. et al. (1978): Mutat. Res. 53, 200

UNEP PUBLICATIONS179OECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(104)

Kawachi, T. et al. (1980): Appl. Methods Oncol. 3, 253 - 267(105)

von der Hude, W. et al. (1988): Mutat. Res. 203, 81 - 94

(106)

Abe, S., Sasaki, M. (1977): J. Natl. Cancer Inst. 58, 1635 - 1640

(107)

Ishidate, M. et al. (1988): Mutat. Res. 195, 151 - 213(108)

Ishidate, M., Odashima, S. (1977): Mutat. Res. 48, 337 - 354(109)

Maxwell, W.A. and Newell, G.W. (1974): Screening techniques for environmental
mutagens. In: Molecular and Environmental Aspects of Mutagenesis (Prakash, L.,
Sherman, F., Lawrence, C.W. & Taber, H.W., eds.), Charles C. Thomas Publisher,
Springfield, Illinois/USA; Chapter 14, pages 223-252(110)

Newell G.W., Maxwell W.A. (1972) Stanford Research Institute, Project LSU-1348 for FDA
U.S. Dept. of Commerce, Services, NTIS PB-221827(111)

Patterson, R.M. et al. (1987): Toxic. in vitro 1, 55 - 57

(112)

Paschin, Y.V. & Bahitova, L.M. (1984): Mutation Res. 137, 57-59(113)

McGregor, D.B. et al. (1988): Environ. Mol. Mutagen. 11, 91 - 118(114)

Sakai, A., Sato, M. (1989): Mutat. Res. 214, 285 - 296(115)

Tokyo Metropolitan Research Laboratory of Public Health (1978):
In: Shell Oil Co. (1992): NTIS/OTS 0535892(116)

Kaul, B.L. (1979): Mutat. Res. 67, 239 - 247(117)

Office of Toxic Substances, US EPA (1984): Chemical Hazard Information Profile Draft
Report, Butylated Hydroxytoluene, August 13, 1984

(118)

Grillo, C.A. & Dulout, F.N., Mutation Research 375, 83-89 (1997)(119)

Bruce, W.R., Heddle, J.A. (1979): Can. J. Genet. Cytol. 21, 319 - 334(120)

Cumming, R.B. et al. (1986): ORNL Biology Div. Annu. Rept.,
ORNL-5195, June 30, 20-22(121)

Paschin et al. (1986): Food Chem. Toxicol. 24, 881-883(122)

Commission Directive of 18 November 1987 adapting to technical progress for the ninth
time Council Directive 67/548/EEC on the approximation of laws, regulations andadministrative provisions relating to the classification, packaging and labelling of dangerous
substances. Official Journal of the European Communities 31, 76 - 78.

(123)

Dean, B.J. (ed.) (1983): Report of the United Kingdom Environmental Mutagen Society(UKEMS) subcommittee on guidelines for mutagenicity testing. Part 1: Basic test
battery; minimal criteria; professional standards; interpretation; selection of supplementary
assays. Chapter 7: Dominant lethal mutation assays, 143 - 164.

(124)

OECD (1984) Guideline 478 "Genetic toxicology: rodent dominant lethal test". OECD
Guidelines for Testing of Chemicals.

(125)

Sheu, C.W. et al. (1986): Environ. Mutagen. 8, 357 - 367(126)

Stanford Research Institute (1977): NTIS/PB 278026

180

UNEP PUBLICATIONSOECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(127)

Tokyo Metropolitan Research Laboratory of Public Health (undated): Chronic toxicity,
teratogenicity and mutagenicity tests with Dibutyl hydroxy toluene; Cytogenetic
studies and dominant lethal test in mice and rats afterlong-term administration of
dibutylhydroxytoluene (BHT) (no further bibliographic data)(128)

Epstein, S.S. et al. (1972): Toxicol. Appl. Pharmacol. 23, 288 - 325(129)

Uno, Y. et al., Mutation Research 320, 189-205 (1994)(130)

Kitchin, K.T. & Brown J.L., Toxicology 88, 31-49 (1994)(131)

Sato, H. et al. (1987): Cancer Lett. 38, 49 - 56

(132)

Singletary, K.W. et al. (1992): Food Chem. Toxicol. 30, 455 - 465(133)

National Cancer Institute (1979): NTIS/PB 298539(134)

Takahashi, M. et al. (1986): Cancer Lett. 30, 161 - 168(135)

Thornton, M. et al. (1989): Carcinogenesis 10, 407 - 410(136)

Preat, V. et al. (1986): Carcinogenesis 7, 1025 - 1028

(137)

Fukushima, S. et al. (1987): Cancer Lett. 34, 83 - 90

(138)

Hagiwara, A. et al. (1989): J. Toxicol. Pathol. 2, 33 - 39

(139)

Williams, G.M. et al. (1991): Cancer Res. 51, 6224 - 6230

(140)

Fukushima, S. et al. (1987): Cancer Res. 47, 2113 - 2116

(141)

Imaida, K. et al. (1988): Cancer Lett. 43, 167 - 172(142)

McCormick, D.L. et al. (1986): Cancer Res. 46, 5264 - 5269

(143)

Cohen, L.A. et al. (1986): J. Natl. Cancer Inst. 76, 721 - 730(144)

Shirai, T. et al. (1991): Carcinogenesis 12, 1335 - 1339

(145)

Thamavit, W. et al. (1989): Cancer Lett. 45, 93 - 101(146)

Hasegawa, R. et al. (1990): Jpn. J. Cancer Res. 81, 871 - 877(147)

Haesen, S. et al. (1988): Carcinogenesis 9, 1755 - 1761

(148)

Hirose, M. et al. (1993): Carcinogenesis 14, 2359 - 2364

(149)

Hirose, M. et al. (1981) Food Cosmet. Toxicol. 19, 147-151(150)

Lindenschmidt, R.C. et al. (1986): Toxicology 38, 151 - 160(151)

Clapp, N.K. et al. (1978): J. Natl. Cancer Inst. 61, 177 - 182(152)

Tokumo, K. et al. (1991): Cancer Lett. 59, 193 - 199(153)

Shirai, T. et al. (1982): Food Chem. Toxicol. 20, 861 - 865(154)

Brooks, T.M. et al. (Sittingbourne Research Centre, Shell Research Ltd.) (1976):
In: Shell Oil Co. (1992): NTIS/OTS 0539579

UNEP PUBLICATIONS

181

OECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(155)

Hinton, R.H. et al. (1990): Toxicologist 10, 297(156)

Robens Institute of Health and Safety (1989): Dose ranging experiment on the role ofhepatocellular injury in the chronic toxicity of BHT, final report 7/88/TX, 20.10.89(157)

Robens Institute of Health and Safety (1990): Long-term toxicity study, effects produced by
BHT in a two generation study - an overview of results obtained to date.

(158)

Robens Institute of Health and Safety (1990): The role of hepatocellular injury in thechronic toxicity of BHT -results to date (February 1990), RI 90/0303, interim report
7/88/TX, 03 April 1990

(159)

Tanaka, T. et al. (1993): Toxicol. Lett. 66, 295 - 304(160)

Han, S.Y. et al. (1993): Teratology 48, 507, B-39

(161)

Tanaka, S. et al. (1990): Eisei Shikenjo Hokoku 108, 52-57(162)

Brown, W.D. et al. (1959) Aust. J. Exp. Biol. 37, 533-548(163)

Smith, S.B. et al., Teratog. Carcinog. Mutagen. 8, 175-189 (1998)(164)

Verhagen, H. (1990): Pharm. Weekbl. Sci. Ed. 12, 164 - 166(165)

Verhagen, H. et al. (1989): Food. Chem. Toxicol. 27, 765 - 772(166)

Shlian, D.M., Goldstone, J. (1986): N. Engl. J. Med. 314, 648 - 649(167)

Grogan, M.W.A. (1986): West. J. Med. 145, 245 - 246(168)

Collings, A.J., Sharratt, M. (1970): Food Cosmet. Toxicol. 8, 409 - 412(169)

Conacher, H.B.S. et al. (1986): Food Chem. Toxicol. 24, 1159 - 1162

(170)

Kirkpatrick, D.C., Lauer, B.H. (1986): Food Chem. Toxicol. 24, 1035 - 1037

(171)

Mizutani, T., Ohe, T. (1976): Eisei Kagaku 22, 265 - 270(172)

Toyoda, M. et al. (1983): J. Jpn. Soc. Nutr. Food Sci. 36, 85 - 96

(173)

de Boer, E.M. et al. (1989): Contact Dermatitis 20, 280 - 286(174)

Goodman, D.L. et al. (1990): J. Allergy Clin. Immunol. 86, 570 - 575(175)

Nunn, C.J. (1991): Food Chem. Toxicol. 29, 73 - 74

(176)

Verhagen, H. et al. (1990): Food Chem. Toxicol. 28,
215 - 220

(177)

Verhagen, H., Kleinjans, J.C.S. (1991): Food Chem. Toxicol. 29, 74 - 75

(178)

WHO (1991): Evaluation of certain food additives and contaminants, Thirty-seventh reportof the Joint FAO/WHO Expert Committee on Food Additives (JEPCA), WHO Technical
Report Series 806, 7 - 8, 41 - 44

(179)

WHO Food Additives Series 42, 429-440 (1999)(180)

Botterweck, A.A.M. et al. (2000): Food Chem. Toxicol. 38, 599-605(181)

Peiler, D. et al., Dermatol. Beruf Umwelt 48, 19-20 (2000)

182

UNEP PUBLICATIONSOECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(182)

Peiler, D. et al., Dermatol. Beruf Umwelt 48, 48-54 (2000)(183)

Kanerva, L. et al. (1999): Acta Derm. Venereol 79, 296-300(184)

Kanerva, L. et al. (1997): Cont. Derm. 37, 301-302(185)

Clapp, N.K. et al. (1974): Food Chem. Toxicol. 12, 367 - 371(186)

Klein, A. & Bruser, B., Life Sciences 50, 883-889 (1992)(187)

Manson,, M.M. et al., Carcinog. 18, 1729-1738 (1997)(188)

Reed, M., Thompson, D.C., Chem. Res. Toxicol. 10, 1109-1117 (1997)(189)

Verschoyle, R.D. et al., Xenobiotica 27, 853-864 (1997)(190)

Jones, P.A. et al., Toxicol. in Vitro 12, 373-382 (1998)(191)

Takahashi, O. (1988) Arch. Toxicol. 62, 325-327(192)

Blumenthal, E.J., Malkinson, A.M. (1987): Arch. Biochem. Biophys. 256, 19 - 28

(193)

Mizutani, T. et al. (1987): Toxicol. Appl. Pharmacol. 87, 166 - 176(194)

Nakagawa, Y, Tayama, K. (1988): Arch. Toxicol. 61, 359 - 365(195)

Nakagawa, Y. (1987): Toxicol. Lett. 37, 251 - 256(196)

Hewitt, S.D. et al. (1990): Carcinogenesis 11, 371 - 375(197)

Andreoli, T. et al. (1991): Toxic. in Vitro 5, 549 - 553(198)

Babich, H., Borenfreund, E. (1990): J. Pharm. Sci. 79, 592 - 594(199)

Bolton, J.L. et al. (1993): Toxicol. Appl. Pharmacol. 123, 43 - 49

(200)

Thompson, J.A. et al. (1991): Exp. Lung Res. 17, 439 - 453(201)

Thompson, J.A. et al. (1991): Adv. Exp. Med. Biol. 283, 393 - 398(202)

Courtheoux, S.I. et al. (1985): Dermatology 6, 153 - 164(203)

Archer, D.L. et al. (1978): Int. Archs. Allergy Appl. Immun. 56, 90 - 93

(204)

Thompson, J.A. et al. (1987): Drug Metabol. Dispos. 5, 833 - 840(205)

Witschi, H. et al. (1989): Pharmac. Ther. 42, 89 - 113(206)

Siegel, D. et al. (1988): Toxicol. Appl. Pharmacol. 96, 68 - 74

(207)

Takahashi, O. (1988): Arch. Toxicol. 62, 325 - 327(208)

Takahashi, O. (1988): Biochem. Pharmacol. 37, 2857 - 2859

(209)

Thompson, D.C. et al. (1986): Adv. Exp. Med. Biol. 197, 301 - 309(210)

Thompson, D.C., Trush, M.A. (1986). Food. Chem. Toxicol. 24, 1189 - 1195

(211)

BUA Report 219, Ergänzungsbericht, VCH, 2000

UNEP PUBLICATIONS

183

OECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(212)

Vavasour, E.(1996): WHO Food Additives Series 35, 3-86

(213)

Athar, M., Iqbal, M., Carcinog. 19, 1133-1139 (1998)(214)

Callaway, J.K. et al., J. Pharmacol. Toxicol. Methods 39, 155-162 (1998)(215)

Can-Eke, B. et al., Chem. Biol. Interact. 113, 65-77 (1998)(216)

Choi, H.-S. & Moore, D.D., Molecular Endocrinology 7, 1596-1602 (1993)(217)

Ciotti, M. et al., Pharmacogenetics 7, 485-495 (1997)(218)

Kurella, E.G. et al., Cell. Mol. Neurobiol. 19, 133-140 (1999)(219)

Lambert, C. et al., Free Radical Biol. Med. 21, 395-400 (1996)(220)

Munday, J.S. et al., Arterio. Throm. Vascul. Biol. 18, 114-119 (1998)(221)

Terrazos-Luch, J. et al., Proc. West. Pharmacol. Soc. 40, 97-99 (1997)(222)

Tu, Z., Anders, M.W., Biochem. Biophys. Res. Comm. 244, 801-805 (1998)(223)

Wanasundara, U.N., Shahidi, F., Fd. Chem. 63, 335-342 (1998)(224)

Davies, R. et al., Carcinogenesis 14, 47-52 (1993)(225)

Burns, C.P. & Petersen, E.S., Journal of Cellular Physiology 144, 36-41 (1990)(226)

Dwight, J.F.St.J. & Hendry B.M., Clinica Chimica Acta 249, 167-181 (1996)(227)

Taniguchi, S. et al., Journal of Dermatological Science 12, 44-49 (1996)(228)

Nakagawa, Y. et al., Bull. Environ. Contam. Toxicol. 52, 511-515 (1994)(229)

Sun, B. et al., Food and Chemical Toxicology 34, 595-601 (1996)(230)

Sun, B. & Fukuhara, M., Toxicology 122, 61-72 (1997)(231)

Budunova, I.V. et al. (1989): Cell. Biol. Toxicol. 5, 77 - 88

(232)

Chauhuri, R. et al. (1993): Cancer Lett. 71, 11 - 18(233)

Tryphonas, H. et al., Food Chem. Toxicol. 37, 671-681 (1999)(234)

Heil, J., Reifferscheid, G. (1992): Carcinogenesis 13, 2389 - 2394

(235)

Richer, N. et al. (1989): Cancer Lett. 47, 211 - 216(236)

Ghosal, A., Iba, M.M. (1992): Mutat. Res. 278, 31 - 41

(237)

Singletary, K.W. (1990): Cancer Lett. 49, 187 - 193(238)

Singletary, K.W., Nelshoppen, J.M. (1991): Carcinogenesis 12, 1967 - 1969(239)

Shibata, M.-A. et al. (1991): Toxicol. Lett. 55, 263 - 272(240)

Daugherty, J.P. et al. (1987): J. Tenn. Acad. Sci. 62, 69 - 71

(241)

Taffe, B.G., Kensler, T.W. (1988): Res. Commun. Chem. Pathol. Pharmacol. 61, 291 - 303

184

UNEP PUBLICATIONSOECD SIDS
6. REFERENCES

2,6-DI-TERT-BUTYL-P-CRESOL (BHT)
ID 128-37-0
DATE: 12.01.2004(242)

Shibata, M.-A. et al. (1989): Toxicol. Appl. Pharmacol. 99, 37 - 49

(243)

Guyton, K.Z. et al. (1991): Proc. Natl. Acad. Sci. USA 88, 946 - 950(244)

Thompson, J.A. et al. (1989): Carcinogenesis 10, 773 - 775(245)

Williams, G.M. et al. (1999): Food Chem. Toxicol. 37, 1027-1038(246)

Walton, K. et al., Food Chem. Toxicol. 37, 1175-1197 (1999)(247)

Kanikkannan, N. et al., Toxicol. Lett. 116, 165-170 (2000)(248)

Lok, E. et al., Carcinogenesis 16, 1071-1078 (1995)(249)

Malkinson, A.M. et al., Cancer Research 57, 2832-2834 (1997)(250)

Matzinger, St.A. et al., Molecular Carcinogenesis 11, 42-48 (1994)(251)

Malkinson, A.M. et al., Cancer Resaerch 57, 2832-2834 (1997)(252)

Miller, A.C.K. et al., Toxicology 90, 141-159 (1994)(253)

Guan, X. et al., Carcinogenesis 16, 2575-2582 (1995)(254)

Cottrell, S. et al., Comp. Haematol Int. 4, 102-107 (1994)(255)

Yamazaki, H. et al., Chemosphere 29, 1293-1299 (1994)(256)

Hirose, M. et al., Food Chem. Toxicol. 37, 985-992 (1999)(257)

Gressani, K.M. et al., Carcinogenesis 20, 2159-2165 (1999)(258)

Dwyer-Nield, L.D. et al., Toxicol. 130, 115-127 (1998)(259)

Umemura, T., Cancer Lett. 145, 101-106 (1999)(260)

Stenius, U. et al., Toxicol. in Vitro 12, 279-285 (1998)(261)

Nagai, F. et al. (1993): Arch. Toxicol. 67, 552 - 557(262)

Schoonderwoerd, S.A. et al. (1991): Arch. Toxicol. 65, 490 - 494(263)

Schrader, T.J., Cooke, G.M., Toxicol. Sci. 53, 278-288 (2000)(264)

Schwade, L.S., Thompson, D.C., In Vitro Mol. Toxicol. 11, 243-253 (1998)(265)

Zimerson, E. and Bruz, M., Contact Dermatitis 39, 222-226 (1998)

UNEP PUBLICATIONS

185